Inhibition of AXL signaling in anti-metastatic therapy

ABSTRACT

Compositions and methods are provided for alleviating cancer in a mammal by administering a therapeutic dose of a pharmaceutical composition that inhibits activity of AXL protein activity, for example by competitive or non-competitive inhibition of the binding interaction between AXL and its ligand GAS6.

The present invention is a divisional of U.S. patent application Ser.No. 13/554,954, filed Jul. 20, 2012, now issued U.S. Pat. No. 8,618,254,which is a continuation of International Patent Application No.PCT/US2011/22125 filed on Jan. 21, 2011, which claims priority from U.S.Provisional Application No. 61/336,478 filed on Jan. 22, 2010, thecontents of which are expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to tumor invasion and metastasis, e.g.,treatments or diagnoses of tumor invasion or metastasis via pathwaysrelated to AXL and/or GAS6

BACKGROUND OF THE INVENTION

Invasion and metastasis are the most insidious and life-threateningaspects of cancer. While tumors with minimal or no invasion may besuccessfully removed, once the neoplasm becomes invasive, it candisseminate via the lymphatics and/or vascular channels to multiplesites, and complete removal becomes very difficult. Invasion andmetastases kill hosts through two processes: local invasion and distantorgan colonization and injury. Local invasion can compromise thefunction of involved tissues by local compression, local destruction, orprevention of normal organ function. The most significant turning pointin cancer, however, is the establishment of distant metastasis. Thepatient can no longer be cured by local therapy alone at this point.

The process of metastasis is a cascade of linked sequential stepsinvolving multiple host-tumor interactions. This complex processrequires the cells to enter into the vascular or lymphatic circulation,arrest at a distant vascular or lymphatic bed, actively extravasate intothe organ interstitium and parenchyma, and proliferate as a secondarycolony. Metastatic potential is influenced by the localmicroenvironment, angiogenesis, stroma-tumor interactions, elaborationof cytokines by the local tissue, and by the molecular phenotype of thetumor and host cells.

Local microinvasion can occur early, even though distant disseminationmay not be evident or may not yet have begun. Tumor cells penetrate theepithelial basement membrane and enter the underlying interstitialstroma during the transition from in situ to invasive carcinoma. Oncethe tumor cells invade the underlying stroma, they gain access to thelymphatics and blood vessels for distant dissemination while releasingmatrix fragments and growth factors. General and widespread changesoccur in the organization, distribution, and quantity of the epithelialbasement membrane during the transition from benign to invasivecarcinoma.

Therapeutic efforts in cancer prevention and treatment are being focusedat the level of signaling pathways or selective modulatory proteins.Protein kinase activities, calcium homeostasis, and oncoproteinactivation are driving signals and therefore may be key regulatory sitesfor therapeutic intervention. Kinases in signaling pathways regulatinginvasion and angiogenesis may be important regulators of metastasis. Oneof the largest classes of biochemical molecular targets is the family ofreceptor tyrosine kinases (RTKs). The most common receptor tyrosinekinase molecular targets to date are the EGF and vascular endothelialgrowth factor (VEGF) receptors. Newer kinase molecular targets includethe type III RTK family of c-kit, and abl. Inhibitors of these moleculeshave been administered in combination with classic chemotherapeutics.

Metastases ultimately are responsible for much of the suffering andmortality from cancer. A need exists to identify and target molecularand functional markers that identify metastatic cancer cells and togenerate reagents for their specific inhibition.

Publications in this field include, inter alia, Li et al. Oncogene.(2009) 28(39):3442-55; United States Patent Application, 20050186571 byUllrich et al.; United States Patent Application 20080293733 by Bearsset al.; Sun et al. Oncology. 2004; 66(6):450-7; Gustafsson et al. ClinCancer Res. (2009) 15(14):4742-9; Wimmel et al. Eur J. Cancer. 200137(17):2264-74; Koorstra et al. Cancer Biol Ther. 2009 8(7):618-26; Taiet al. Oncogene. (2008) 27(29):4044-55

The receptor tyrosine kinase AXL (also known as Ufo and Tyro7) belongsto a family of tyrosine receptors that includes Tyro3 (Sky) and Mer(Tyro12). A common ligand for AXL family is GAS6 (Growth arrest-specificprotein 6). Human AXL is a 2,682-bp open reading frame capable ofdirecting the synthesis of an 894-amino acid polypeptide. Two variantmRNAs have been characterized, transcript variant 1 may be accessed atGenbank, NM_(—)021913.3 and transcript variant 2 may be accessed atNM_(—)001699.4. The polypeptide sequence of the native protein isprovided as SEQ ID NO:1, and specific reference may be made to thesequence with respect to amino acid modifications. Important cellularfunctions of GAS6/AXL include cell adhesion, migration, phagocytosis,and inhibition of apoptosis. GAS6 and AXL family receptors are highlyregulated in a tissue and disease specific manner.

AXL is characterized by a unique molecular structure, in that theintracellular region has the typical structure of a receptor tyrosinekinase and the extracellular domain contains fibronectin III and Igmotifs similar to cadherin-type adhesion molecules. During development,AXL is expressed in various organs, including the brain, suggesting thatthis RTK is involved in mesenchymal and neural development. In theadult, AXL expression is low but returns to high expression levels in avariety of tumors. GAS6 is, so far, the single, activating ligand forAXL.

Receptor tyrosine kinases (RTK) are generally activated by ligands thatpromote receptor dimerisation and, in turn, autophosphorylation oftyrosine residues within the cytosolic domain. Binding of signalingproteins to these phosphorylated tyrosine residues then leads todownstream signaling. AXL family RTKs are unique in that they areactivated by GAS6, a member of the vitamin K-dependent protein familythat resembles blood coagulation factors rather than typical growthfactors.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that AXL and/orGAS6 related pathways are related to tumor invasion and/or metastasis.Accordingly, the present invention provides compositions and methodsuseful for treating tumor invasion and/or metastasis, e.g., viainhibition of AXL and/or GAS6 related pathways. In addition, the presentinvention provides reagents and methods useful for determining thesusceptibility or likelihood of a tumor to become invasive and/ormetastatic, e.g., via detecting the level of activity of AXL and/orGAS6.

In one embodiment, the present invention provides soluble AXL variantpolypeptides, wherein said polypeptide lacks the AXL transmembranedomain, and optionally intracellular domain and comprises at least oneamino acid modification relative to the wild-type AXL sequence, andwherein said change increases the affinity of the AXL polypeptidebinding to GAS6. In some embodiments, the soluble AXL variantpolypeptide comprises at least one amino acid modification within aregion selected from the group consisting of 1) between 15-50, 2)between 60-120, and 3) between 125-135 of the wild-type AXL sequence(SEQ ID NO: 1). In some other embodiments, the soluble AXL variantpolypeptide comprises at least one amino acid modification at position19, 23, 26, 27, 32, 33, 38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90,92, 97, 98, 105, 109, 112, 113, 116, 118, 127 or 129 of the wild-typeAXL sequence (SEQ ID NO: 1) or a combination thereof. In some otherembodiments, the soluble AXL variant polypeptide comprises at least oneamino acid modification selected from the group consisting of 1) A19T,2) T23M, 3) E26G, 4) E27G or E27K, 5) G32S, 6) N33S, 7) T38I, 8) T44A,9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E, 14) V79M, 15) Q86R, 16)D87G, 17) D88N, 18) I90M or I90V, 19) V92A, V92G or V92D, 20) I97R, 21)T98A or T98P, 22) T105M, 23) Q109R, 24) V112A, 25) F113L, 26) H116R, 27)T118A, 28) G127R or G127E, and 29) E129K and combinations andconservative equivalents thereof.

In yet some other embodiments, the soluble AXL variant polypeptidecomprises amino acid changes relative to the wild-type AXL sequence (SEQID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid87; (c) valine 92; and (d) glycine 127. In yet some other embodiments,the soluble AXL variant polypeptide contains glycine 32 residue replacedwith a serine residue, aspartic acid 87 residue replaced with a glycineresidue, valine 92 residue replaced with an alanine residue, or glycine127 residue replaced with an arginine residue or a combination orconservative equivalent thereof. In still some other embodiments, thesoluble AXL variant polypeptide comprises amino acid changes relative tothe wild-type AXL sequence (SEQ ID NO: 1) at the following positions:(a) glutamic acid 26; (b) valine 79; (c) valine 92; and (d) glycine 127.In still some other embodiments, the soluble AXL variant polypeptidecontains glutamic acid 26 residue replaced with a glycine residue,valine 79 residue replaced with a methionine residue, valine 92 residuereplaced with an alanine residue, or glycine 127 residue replaced with aglutamic acid residue or a combination or conversative equivalentthereof.

In still yet some other embodiments, the soluble AXL variant polypeptidecomprises at least amino acids 1-437, 19-437, 130-437, 19-132, 1-132 ofthe wild-type AXL polypeptide (SEQ ID NO: 1). In still yet some otherembodiments, the soluble AXL variant polypeptide is a fusion proteincomprising an Fc domain.

In one embodiment, the soluble AXL variant polypeptide has an affinityof at least about 1×10⁻⁵ M for GAS6. In another embodiment, the solubleAXL variant polypeptide has an affinity of at least about 1×10⁻⁶ M, forGAS6. In yet another embodiment, the soluble AXL variant polypeptide hasan affinity of at least about 1×10⁻⁷ M for GAS6. In yet anotherembodiment, the soluble AXL variant polypeptide has an affinity of atleast about 1×10⁻⁸ M for GAS6. In yet another embodiment, the solubleAXL variant polypeptide has an affinity of at least about 1×10⁻⁹ M,1×10⁻¹⁰M, 1×10⁻¹¹M, or 1×10⁻¹²M for GAS6. In various embodimentsdescribed herein, the soluble AXL variant polypeptide exhibits anaffinity to GAS6 that is at least about 2-fold stronger than theaffinity of the wild-type AXL polypeptide. In some embodiments, thesoluble AXL variant polypeptide exhibits an affinity to GAS6 that is atleast about 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold,or 30-fold stronger than the affinity of the wild-type AXL polypeptide.

In another embodiment, the present invention provides isolatedantibodies or fragments thereof which specifically bind to a GAS6protein (SEQ ID NO: 2). In some embodiments, the isolated antibody orfragment thereof is a monoclonal antibody, a humanized antibody, achimeric antibody, a single chain antibody (ScFv), or a combinationthereof. In some other embodiments, the isolated antibody or fragmentthereof binds an epitope comprised in one or more amino acid regions ofGAS6 selected from the group consisting of R299-T317, V364-P372,R389-N396, D398-A406, E413-H429, and W450-M468. In yet some otherembodiments, the isolated antibody or fragment thereof binds an epitopecomprised in the amino acid region selected from the group consisting ofRMFSGTPVIRLRFKRLQPT (SEQ ID NO: 3), VGRVTSSGP (SEQ ID NO: 4), RNLVIKVN(SEQ ID NO: 5), DAVMKIAVA (SEQ ID NO: 6), ERGLYHLNLTVGGIPFH (SEQ ID NO:7), and WLNGEDTTIQETVKVNTRM (SEQ ID NO: 8).

In yet another embodiment, the present invention provides methods oftreating, reducing, or preventing the metastasis or invasion of a tumorin a mammalian patient. In one embodiment, the method comprisesadministering to said patient an effective dose of a soluble AXL variantpolypeptide or an isolated anti-GAS6 antibody or fragment thereof.

In still another embodiment, the present invention provides methods oftreating, reducing, or preventing the metastasis or invasion of a tumorin a mammalian patient. In one embodiment, the method comprisesadministering one or more inhibitors selected from the group consistingof (a) an inhibitor of AXL activity (b) an inhibitor of GAS6 activity;and (c) an inhibitor of AXL-GAS6 interaction. In various embodimentsdescribed herein, the inhibitor is a polypeptide, a polynucleotide, asmall molecule, an antibody, an antibody fragment, or antibodydrug-conjugate.

In still yet another embodiment, the present invention provides methodsof determining the ability of a tumor to undergo invasion or metastasisin a subject. In one embodiment, the method comprises detecting thelevel of AXL activity and/or GAS6 activity in a biological sample from asubject with a tumor; and comparing the level of the AXL and/or GAS6activity in the biological sample to predetermined level, wherein anincrease over the predetermined level is indicative of a predispositionof the tumor to invasion or metastasize.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. AXL expression correlates tumor progression and metastasis inhuman breast and ovarian cancer. A. Representative images of AXLimmunohistochemical staining in normal breast tissue (normal), primaryinfiltrating ductal carcinoma (grade I, 2, and 3) and lymph nodemetastases (lymph node). Note that high levels of membranous AXLstaining were present a grade 2 (arrows), grade 3, and lymph nodemetastases. No AXL staining was observed in normal or tumor stroma (*).B. Representative images of AXL immunohistochemical staining in normalovarian epithelium (arrow). stage II, stage III, and omentum metastasisderived from patients with serous adenocarcinoma. Note that normal andtumor stroma were negative for AXL staining (*).

FIG. 2. Genetic inactivation of AXL is sufficient to block breast andovarian metastasis. A. H&E and AXL immunohistochemical staining in thelungs of mice tail vein injected with shscramble (shSCRM) and shAXL(shAXL) MDA-231 cells. Photographs are representative of 5 mice pergroup. Graphs depict real time PCR analysis of human GASPDH and AXLexpression in whole lung from mice injected with shSCRM or shAXL MDA-231cells (n=5) B. Photographs of mice taken 28 days after injection withshscramble (shSCRM) and shAXL (shAXL) SKOV3ip.1 cells. Note that theshSCRM injected mice developed numerous metastases in throughout theabdominal cavity (circled). For the shAXL group, the mouse with thegreatest tumor burden is shown. Graphs to the right depict the averagenumber of peritoneal metastases per mouse >5 mm in size and the averageweight of the largest tumor. Photographs are representative of 5 miceper group. C. Photographs of mice taken 34 days after injection withshSCRM and shAXL OVCAR-8 cells. Note that the shSCRM injected micedeveloped numerous metastases in throughout the abdominal cavity(circled). Graphs to the right depict the average total number ofperitoneal metastases per mouse and the average total tumor weight.Photographs are representative of 8 mice per group.

FIG. 3. Genetic inactivation of AXL does not affect breast or ovariantumor cell proliferation in vitro or growth in vivo. A. Cellular growthcurves for MDA-231, SKOV3ip.1, and OVCAR-8 cells stably expressing shRNAtargeting sequences for scramble control (shSCRM) or AXL (shAXL).Measurements were performed in triplicate and error bars represent theS.E.M. B. Average tumor volumes of orthotopic MDA-231 (n=8 mice pergroup) and subcutaneous SKOV3ip.1 tumors (n=4 mice per group) grown overa 48-day time course. Error bars represent the S.E.M.

FIG. 4. AXL regulates ovarian and breast tumor cell invasion in vitro.A. Collagen invasion assay of control (shSCRM) and AXL deficient (shAXL)MDA-231, SKOV3ip.1, and OVCAR-8 cells. Photographs are representative of3 samples per group and were taken 7 days after plating cells incollagen. Note the invasive phenotype observed in AXL wild-type cells(branching) compared to AXL deficient cells (rounded). Graphs showquantification of collagen invasion assays. B. Real time PCR analysis ofMMP-2 expression in shAXL and shSCRM SKOV3ip.1 cells. Expression valueswere normalized to 18S; n=3. Error bars represent the S.E.M. Asterisksindicate a significant increase or decrease in expression compared toshSCRM as determined by the student's t-test (**, P<0.001). C. MMP-2reporter assay of shSCRM or shAXL SKOV3ip.1 cells (n=6). D. Gelatinzymography assay for pro- and active-MMP2 activity in conditioned mediacollected from serum starved SKOV3ip.1 cells. E. Western blot analysisof phospho-AKT at Ser473 (P-AKT), total AKT (AKT), and AXL expression inSKOV3ip.1 cells expressing shRNA sequences targeting scramble control(shSCRM) or AXL (shAXL) and starved SKOV3ip.1 cells (strve) treated withGAS6 or the PI3K inhibitor Ly294002 (Ly) with GAS6. F. MMP-2 reporterassay in starved SKOV3ip.1 cells (strve) treated with GAS6 or GAS6 withthe PI3K inhibitor Ly294002 (Ly+GAS6).

FIG. 5. Soluble AXL ectodomain therapy inhibits AXL signaling andinvasion in vitro. A. Schematic representation of the mechanism forsoluble AXL therapy. Soluble AXL (sAXL) functions as a decoy receptor toinhibit endogenous AXL signaling. B. Western blot analysis ofphospho-AKT at Ser473 (P-AKT), total AKT (AKT), and AXL expression inMDA231, SKOV3ip.1, and OVCAR-8 cells expressing shRNA sequencestargeting scramble control (shSCRM) or AXL (shAXL) and starved SKOV3ip.1cells (strve) treated with GAS6 or the PI3K inhibitor Ly294002 (Ly) withGAS6. C. Western blot analysis of phospho-AKT Ser473 expression in cellstreated with conditioned media containing the soluble AXL receptor(sAXL) or control media (−). All cells were starved for 48 hours andtreated with GAS6 (+) or vehicle (−). D. Collagen invasion assay inMDA-231 cells treated with conditioned media containing control vectoror sAXL.

FIG. 6. Treatment with soluble AXL receptors inhibits metastatic tumorburden in mice with established metastases. A. Schematic representationof the soluble AXL receptor treatment study. Nude mice were i.p.injected with 1×10⁶ SKOV3ip.1 cells. Five days after implantation, thepresence of macroscopic lesions was verified in mice (shown is arepresentative photograph of a mouse with peritoneal metastasis at day 5following injection, metastatic lesions are circled). At day 7, micewere injected with adenoviruses expression the IgG2a-Fc control (Ad-Fc)or soluble AXL receptor (Ad-sAXL). Serum levels of sAXL expression wasassessed by western blot analysis every 3-4 days following adenoviralinjection. Day 28 following tumor cell implantation tumor burden wasassessed in all mice. B. Representative photographs of mice treated withadenoviruses expressing Ad-sAXL or Ad-Fc at 28 days following tumor cellinjection. Metastatic lesions are circled. Graphs show the average totaltumor number and weight for 7 mice per group. Error bars represent theS.E.M. Note that a statistical difference in tumor number and weight(p=0.01, students t test) was observed between Ad-Fc and Ad-sAXL treatedmice (*). C. Real time PCR analysis of MMP-2 expression in tumors ofmice treated with Ad-Fc or Ad-AXL.

FIG. 7. Soluble AXL ectodomain therapy does not induce normal tissuetoxicity. A. Complete CBC and serum chemistry analysis of mice treatedwith control (Fc) or soluble AXL therapy (sAXL). B. H&E staining ofliver and kidney tissue collected from mice treated with Fc or sAXL.

FIG. 8. Schematic diagram illustrating the molecular mechanismsassociated with soluble AXL receptor inhibition of metastasis. SolubleAXL receptor (sAXL) therapy functions as a decoy receptor that binds tothe AXL ligand GAS6. sAXL inhibits endogenous GAS6-AXL signaling eventsthat stimulate cellular invasion and metastasis.

FIG. 9. Generation of AXL deficient breast and ovarian cancer celllines. A. Western blot analysis of AXL expression in a panel of humanbreast and ovarian cancer cell lines. Heat shock protein 70 (Hsp70) wasused as a protein loading control. B. Western blot analysis of AXLexpression in metastatic breast (MDA-231) ovarian (SKOV3ip.1 andOVCAR-8) cancer cell lines stably transfected with shRNA targetingsequences for scramble control (shSCRM) or AXL (shAXL). Note that theshAXL cell lines have a significant reduction in AXL expression.

FIG. 10. AXL does not affect breast and ovarian tumor cell adhesion orsurvival. A-B. Percent cell migration of MDA-231 (A) and SKOV3ip.1 (B)cells in boyden chamber migration assays towards serum as thechemoattractant. C-D. Analysis of MDA-231 (A) SKOV3ip.1 (B) cellularadhesion to extracellular matrix proteins. Abbreviations: bovine serumalbumin (BSA), fibronectin (FN), collagen type I (Col I), collagen typeIV (Col IV), laminin (LN), fibrinogen (FBN). Error bars represent thestandard error of the mean. E-F. Survival analysis of AXL wild-type andAXL deficient MDA-231 (E) and SKOV3ip.1 (F) tumor cells following serumwithdrawal as determined by the XTT assay.

FIG. 11. Treatment with soluble AXL receptors inhibits metastatic tumorburden in mice with established OVCAR-8 metastases. A. Schematicrepresentation of the soluble AXL receptor treatment study. Nude micewere i.p. injected with 5×10⁶ OVCAR-8 cells. Fourteen days afterimplantation, the presence of macroscopic lesions was verified in mice(shown is a representative photograph of a mouse with peritonealmetastasis at day 14 following injection, metastatic lesions arecircled). At day 14, mice were injected with adenoviruses expression theIgG2a-Fc control (Ad-Fc) or soluble AXL receptor (Ad-sAXL). Serum levelsof sAXL expression was assessed by western blot analysis. Day 34following tumor cell implantation tumor burden was assessed in all mice.B. Representative photographs of mice treated with adenovirusesexpressing Ad-sAXL or Ad-Fc at 28 days following tumor cell injection.Metastatic lesions are circled. C. Graphs show the average total tumornumber and weight for 8 mice per group. Error bars represent the S.E.M.Note that a statistical difference in tumor number and weight (p<0.01,students t test) was observed between Ad-Fc and Ad-sAXL treated mice(*).

FIG. 12. Binding of AXL Library sort 5 products to GAS6. Flow cytometrydot plots of yeast cells expressing either wild-type AXL (A) or thepooled AXL Sort 5 products from the directed evolution work (B). Datashows binding following off-rate tests as described in Example 2. Levelsof binding to 2 nM Gas6 are shown in the left column, levels of bindingto Gas6 following a 4 hour unbinding step are shown in the middlecolumn, and levels of binding to Gas6 following a 6 hour unbinding stepare shown in the right column. For cells that are positive forexpression of the particular protein on its cell surface (upper rightquadrant of each flow cytometry dot plot), binding levels to Gas6(y-axis) are quantified in the bar graph below. The pooled Sort 5products show significantly improved Gas6 binding compared to wild-typeAXL.

FIG. 13. Binding of enhanced AXL variants to GAS6. Left panel showsequilibrium binding towards Gas6 by the AXL mutants S6-1 (red squares)and S6-2 (blue diamonds) as compared to wild-type AXL (green circles).The mutants S6-1 and S6-2 exhibit significantly higher levels of bindingto lower concentrations of Gas6, demonstrating stronger binding affinityfor these mutants compared to wild-type AXL. The right panel showsdissociation kinetics of the wild-type or engineered Gas6-AXLinteraction. The wild-type Gas6-AXL interaction (“wild-type”)dissociates rapidly as a function of time, wild the engineeredinteraction between Gas6 and S6-1 (“S6-1”) or S6-2 (“S6-2”) showssignificantly increased retention of binding.

FIG. 14. Intraperitoneal delivery of purified AXL S6-1-Fc shows enhancedtherapeutic effects over wild-type AXL-Fc and AXL E59R/T77R-Fc. Tworepresentative images from the necropsies of mice from three treatmentgroups, AXL E59R/T77R-Fc, wild-type AXL-Fc, and AXL S6-1-Fc, are shown.Black circles indicate metastatic lesions visible in the images, but donot necessarily indicate all metastatic sites. Wild-type AXL-Fc showsmoderate inhibition of metastasis over the negative control, AXLE59R/T77R, while AXL S6-1 shows nearly complete inhibition ofmetastasis.

FIG. 15. Inhibition of metastasis in SKOV3ip.1 xenograph model. In thetop two graphs, the same data set is presented in two different ways toindicate the average number of metastatic lesions counted in eachtreatment group. Similarly, the bottom two graphs show the same data setwhich outlines the total weight of all metastasis excised from mice ineach treatment group. Wild-type AXL-Fc inhibits the spread of metastasisas compared to negative control E59R/T77R-Fc, as indicated by a decreasein both number of lesions (top panel) as well as overall weight (bottompanel). AXL S6-1-Fc shows significant reduction in tumor burden ascompared to both wild-type AXL-Fc and AXL E59R/T77R-Fc as assessed bynumber of lesions (top panel) as well as overall weight (bottom panel).These data demonstrate that the enhanced affinity of AXL S6-1 offersimproved therapeutic efficacy over wild-type and that AXL S6-1-Fc is aviable treatment for the management of metastasis.

DEFINITIONS

In the description that follows, a number of terms conventionally usedin the field of cell culture are utilized extensively. In order toprovide a clear and consistent understanding of the specification andclaims, and the scope to be given to such terms, the followingdefinitions are provided.

“Inhibitors,” “activators,” and “modulators” of AXL on metastatic cellsor its ligand GAS6 are used to refer to inhibitory, activating, ormodulating molecules, respectively, identified using in vitro and invivo assays for receptor or ligand binding or signaling, e.g., ligands,receptors, agonists, antagonists, and their homologs and mimetics.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an .alpha. carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. All single lettersused in the present invention to represent amino acids are usedaccording to recognized amino acid symbols routinely used in the field,e.g., A means Alanine, C means Cysteine, etc. An amino acid isrepresented by a single letter before and after the relevant position toreflect the change from original amino acid (before the position) tochanged amino acid (after position). For example, A19T means that aminoacid alanine at position 19 is changed to threonine.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” thus encompass individuals havingcancer, including without limitation, adenocarcinoma of the ovary orprostate, breast cancer, glioblastoma, etc., including those who haveundergone or are candidates for resection (surgery) to remove canceroustissue. Subjects may be human, but also include other mammals,particularly those mammals useful as laboratory models for humandisease, e.g. mouse, rat, etc.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. In general, cellsof interest for detection, analysis, classification, or treatment in thepresent application include precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and non-metastatic cells. Examples of cancerinclude but are not limited to, ovarian cancer, glioblastoma, breastcancer, colon cancer, lung cancer, prostate cancer, hepatocellularcancer, gastric cancer, pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, cancer of the urinary tract,thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer,and brain cancer.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body. Therefore,the present invention contemplates a method of determining the risk offurther growth of one or more cancerous tumors in an organ or body partwhich is not directly connected to the organ of the original canceroustumor and/or any steps in a process leading up to that growth.

Depending on the nature of the cancer, an appropriate patient sample isobtained. As used herein, the phrase “cancerous tissue sample” refers toany cells obtained from a cancerous tumor. In the case of solid tumorswhich have not metastasized, a tissue sample from the surgically removedtumor will typically be obtained and prepared for testing byconventional techniques.

The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents; washed; orenrichment for certain cell populations, such as cancer cells. Thedefinition also includes sample that have been enriched for particulartypes of molecules, e.g., nucleic acids, polypeptides, etc. The term“biological sample” encompasses a clinical sample, and also includestissue obtained by surgical resection, tissue obtained by biopsy, cellsin culture, cell supernatants, cell lysates, tissue samples, organs,bone marrow, blood, plasma, serum, and the like. A “biological sample”includes a sample obtained from a patient's cancer cell, e.g., a samplecomprising polynucleotides and/or polypeptides that is obtained from apatient's cancer cell (e.g., a cell lysate or other cell extractcomprising polynucleotides and/or polypeptides); and a sample comprisingcancer cells from a patient. A biological sample comprising a cancercell from a patient can also include non-cancerous cells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of breast cancer, prostate cancer,or other type of cancer.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of cancer-attributable death or progression, includingrecurrence, metastatic spread, and drug resistance, of a neoplasticdisease, such as ovarian cancer. The term “prediction” is used herein torefer to the act of foretelling or estimating, based on observation,experience, or scientific reasoning. In one example, a physician maypredict the likelihood that a patient will survive, following surgicalremoval of a primary tumor and/or chemotherapy for a certain period oftime without cancer recurrence.

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure (e.g., radiation,a surgical procedure, etc.), for the purposes of obtaining an effect.The effect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of effecting a partial or complete cure for a disease and/orsymptoms of the disease. “Treatment,” as used herein, covers anytreatment of any metastatic tumor in a mammal, particularly in a human,and includes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease. In tumor (e.g., cancer)treatment, a therapeutic agent may directly decrease the metastasis oftumor cells.

Treating may refer to any indicia of success in the treatment oramelioration or prevention of an cancer, including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the disease condition more tolerable to the patient;slowing in the rate of degeneration or decline; or making the finalpoint of degeneration less debilitating. The treatment or ameliorationof symptoms can be based on objective or subjective parameters;including the results of an examination by a physician. Accordingly, theterm “treating” includes the administration of the compounds or agentsof the present invention to prevent or delay, to alleviate, or to arrestor inhibit development of the symptoms or conditions associated withneoplasia, e.g., tumor or cancer. The term “therapeutic effect” refersto the reduction, elimination, or prevention of the disease, symptoms ofthe disease, or side effects of the disease in the subject.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic and the compounds as used herein. Whenadministered in combination, each component can be administered at thesame time or sequentially in any order at different points in time.Thus, each component can be administered separately but sufficientlyclosely in time so as to provide the desired therapeutic effect.

According to the present invention, the first therapeutic can be anysuitable therapeutic agent, e.g., cytotoxic agents. One exemplary classof cytotoxic agents are chemotherapeutic agents, e.g., they can becombined with treatment to inhibit AXL or GAS6 signaling. Exemplarychemotherapeutic agents include, but are not limited to, aldesleukin,altretamine, amifostine, asparaginase, bleomycin, capecitabine,carboplatin, carmustine, cladribine, cisapride, cisplatin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,docetaxel, doxorubicin, dronabinol, duocarmycin, epoetin alpha,etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine,granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha,irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna,methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone,omeprazole, ondansetron, paclitaxel (Taxol™), pilocarpine,prochloroperazine, rituximab, saproin, tamoxifen, taxol, topotecanhydrochloride, trastuzumab, vinblastine, vincristine and vinorelbinetartrate. For ovarian cancer treatment, a preferred chemotherapeuticagent with which an AXL or GAS6 signaling inhibitor can be combined ispaclitaxel (Taxol™).

Other combination therapies are radiation, surgery, and hormonedeprivation (Kwon et al., Proc. Natl. Acad. Sci U.S.A., 96: 15074-9,1999). Angiogenesis inhibitors can also be combined with the methods ofthe invention.

“Concomitant administration” of a known cancer therapeutic drug with apharmaceutical composition of the present invention means administrationof the drug and AXL inhibitor at such time that both the known drug andthe composition of the present invention will have a therapeutic effect.Such concomitant administration may involve concurrent (i.e. at the sametime), prior, or subsequent administration of the drug with respect tothe administration of a compound of the present invention. A person ofordinary skill in the art would have no difficulty determining theappropriate timing, sequence and dosages of administration forparticular drugs and compositions of the present invention.

As used herein, the phrase “disease-free survival,” refers to the lackof such tumor recurrence and/or spread and the fate of a patient afterdiagnosis, with respect to the effects of the cancer on the life-span ofthe patient. The phrase “overall survival” refers to the fate of thepatient after diagnosis, despite the possibility that the cause of deathin a patient is not directly due to the effects of the cancer. Thephrases, “likelihood of disease-free survival”, “risk of recurrence” andvariants thereof, refer to the probability of tumor recurrence or spreadin a patient subsequent to diagnosis of cancer, wherein the probabilityis determined according to the process of the invention.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters. Also, certain compounds named in thisinvention may be present in more than one stereoisomeric form, and thenaming of such compounds is intended to include all single stereoisomersand all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

A “therapeutically effective amount” means the amount that, whenadministered to a subject for treating a disease, is sufficient toeffect treatment for that disease.

DETAILED DESCRIPTIONS

According to the present invention, it provides soluble AXL variants,e.g., soluble AXL variant polypeptides that have a binding activity toGAS6 that is substantially equal to or better than the binding activityof a wild-type AXL polypeptide. In some embodiments of the invention,the soluble AXL variant polypeptides are utilized as therapeutic agents.

The AXL protein, with reference to the native sequence of SEQ ID NO: 1,comprises an immunoglobulin (Ig)-like domain from residues 27-128, asecond Ig-like domain from residues 139-222, fibronectin type 3 domainsfrom residues 225-332 and 333-427, intracellular domain from residues473-894 including tyrosine kinase domain. The tyrosine residues at 779,821 and 866 become autophosphorylated upon receptor dimerization andserve as docking sites for intracellular signaling molecules. The nativecleavage site to release the soluble form of the polypeptide lies atresidues 437-451.

For the purposes of the invention, a soluble form of AXL is the portionof the polypeptide that is sufficient to bind GAS6 at a recognizableaffinity, e.g., high affinity, which normally lies between the signalsequence and the transmembrane domain, i.e. generally from about SEQ IDNO: 1 residue 19-437, but which may comprise or consist essentially of atruncated version of from about residue 19, 25, 30, 35, 40, 45, 50 toabout residue 132, 450, 440, 430, 420, 410, 400, 375, 350, to 321, e.g.,residue 19-132. In some embodiments, a soluble form of AXL lacks thetransmembrane domain, and optionally the intracellular domain.

Soluble AXL variant polypeptides (sAXL variants) of the presentinvention include one or more amino acid modifications within thesoluble form of wild-type AXL, e.g., one or more amino acidmodifications that increase its affinity for GAS6. According to thepresent invention, amino acid modifications include any naturallyoccurring or man-made amino acid modifications known or later discoveredin the field. In some embodiments, amino acid modifications include anynaturally occurring mutation, e.g., substitution, deletion, addition,insertion, etc. In some other embodiments, amino acid modificationsinclude replacing existing amino acid with another amino acid, e.g., aconservative equivalent thereof. In yet some other embodiments, aminoacid modifications include replacing one or more existing amino acidswith non-natural amino acids or inserting one or more non-natural aminoacids. In still some other embodiments, amino acid modifications includeat least 1, 2, 3, 4, 5, or 6 or 10 amino acid mutations or changes.

In some exemplary embodiments, one or more amino acid modifications canbe used to alter properties of the soluble form of AXL, e.g., affectingthe stability, binding activity and/or specificity, etc. Techniques forin vitro mutagenesis of cloned genes are known. Examples of protocolsfor scanning mutations may be found in Gustin et al., Biotechniques14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al., Mol GenGenet 199:537-9 (1985); and Prentki et al., Gene 29:303-13 (1984).Methods for site specific mutagenesis can be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108;Weiner et al., Gene 126:35-41 (1993); Sayers et al., Biotechniques13:592-6 (1992); Jones and Winistorfer, Biotechniques 12:528-30 (1992);Barton et al., Nucleic Acids Res 18:7349-55 (1990); Marotti and Tomich,Gene Anal Tech 6:67-70 (1989); and Zhu Anal Biochem 177:120-4 (1989).

In some embodiments, sAXL variants of the present invention include oneor more amino acid modifications within one or more regions of residue18 to 130, residue 10 to 135, residue 15 to 45, residue 60 to 65,residue 70 to 80, residue 85 to 90, residue 91 to 99, residue 104 to110, residue 111 to 120, residue 125 to 130, residue 19 to 437, residue130 to 437, residue 19 to 132, residue 21 to 132, residue 21 to 121,residue 26 to 132, or residue 26 to 121 of wild-type AXL (SEQ ID NO: 1).In some other embodiments, sAXL variants of the present inventioninclude one or more amino acid modifications within one or more regionsof residue 20 to 130, residue 37 to 124 or residue 141 to 212 ofwild-type AXL (SEQ ID NO: 1). In yet some other embodiments, sAXLvariants of the present invention include one or more amino acidmodifications at one or more positions of position 19, 23, 26, 27, 32,33, 38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105,109, 112, 113, 116, 118, 127, or 129 of wild-type AXL (SEQ ID NO: 1).

In yet some other embodiments, sAXL variants of the present inventioninclude one or more amino acid modifications including without anylimitation 1) A19T, 2) T23M, 3) E26G, 4) E27G or E27K, 5) G32S, 6) N33S,7) T38I, 8) T44A, 9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E, 14)V79M, 15) Q86R, 16) D87G, 17) D88N, 18) I90M or I90V, 19) V92A, V92G orV92D, 20) I97R, 21) T98A or T98P, 22) T105M, 23) Q109R, 24) V112A, 25)F113L, 26) H116R, 27) T118A, 28) G127R or G127E, and 29) E129K and acombination thereof.

In yet some other embodiments, sAXL variants of the present inventioninclude one or more amino acid modifications at position 32, 87, 92, or127 of wild-type AXL (SEQ ID NO: 1) or a combination thereof, e.g.,G32S; D87G; V92A and/or G127R. In yet some other embodiments, sAXLvariants of the present invention include one or more amino acidmodifications at position 26, 79, 92, 127 of wild-type AXL (SEQ IDNO: 1) or a combination thereof, e.g., E26G, V79M; V92A and/or G127E.

According to the present invention, sAXL variants of the presentinvention can be further modified, e.g., joined to a wide variety ofother oligopeptides or proteins for a variety of purposes. For instance,various post-translation or post-expression modifications can be carriedout with respect to sAXL variants of the present invention. For example,by employing the appropriate coding sequences, one may providefarnesylation or prenylation. In some embodiments, the sAXL variants ofthe present invention can be PEGylated, where the polyethyleneoxy groupprovides for enhanced lifetime in the blood stream. The sAXL variants ofthe present invention can also be combined with other proteins, such asthe Fc of an IgG isotype, which can be complement binding, with a toxin,such as ricin, abrin, diphtheria toxin, or the like, or with specificbinding agents that allow targeting to specific moieties on a targetcell.

In some embodiments, sAXL variants of the present invention is a fusionprotein, e.g., fused in frame with a second polypeptide. In someembodiments, the second polypeptide is capable of increasing the size ofthe fusion protein, e.g., so that the fusion protein will not be clearedfrom the circulation rapidly. In some other embodiments, the secondpolypeptide is part or whole of Fc region. In some other embodiments,the second polypeptide is any suitable polypeptide that is substantiallysimilar to Fc, e.g., providing increased size and/or additional bindingor interaction with Ig molecules. In yet some other embodiments, thesecond polypeptide is part or whole of an albumin protein, e.g., a humanserum albumin protein.

In some other embodiments, the second polypeptide is useful for handlingsAXL variants, e.g., purification of sAXL variants or for increasing itsstability in vitro or in vivo. For example, sAXL variants of the presentinvention can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric or fusion polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. EPA 394,827; Traunecker et al., Nature, 331:84-86, 1988. Fusion proteins having disulfide-linked dimeric structures(due to the IgG) can also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

In yet some other embodiments, the second polypeptide is a markersequence, such as a peptide which facilitates purification of the fusedpolypeptide. For example, the marker amino acid sequence can be ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Another peptide tag useful for purification, the “HA” tag,corresponds to an epitope derived from the influenza hemagglutininprotein. Wilson et al., Cell 37: 767, 1984.

In still some other embodiments, the second polypeptide is an entityuseful for improving the characteristics of sAXL variants of the presentinvention. For instance, a region of additional amino acids,particularly charged amino acids, may be added to the N-terminus of thepolypeptide to improve stability and persistence during purificationfrom the host cell or subsequent handling and storage. Also, peptidemoieties may be added to the sAXL variants of the present invention tofacilitate purification and subsequently removed prior to finalpreparation of the polypeptide. The addition of peptide moieties tofacilitate handling of polypeptides are familiar and routine techniquesin the art.

In still yet some embodiments, sAXL variants of the present inventionhas a binding activity to GAS6 that is at least equal or better than thewild-type AXL. In some other embodiments, sAXL variants of the presentinvention has a binding activity or affinity to GAS6 that is at least1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold greater than that ofthe wild-type AXL. In some other embodiments, sAXL variants of thepresent invention has a binding activity or affinity to GAS6 of at leastabout 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸ or 1×10⁻⁸ M. In yet some other embodiments,sAXL variants of the present invention is capable of inhibiting, inhibitor compete with wild-type AXL binding to GAS6 either in vivo, in vitroor both. In yet some other embodiments, sAXL variants of the presentinvention inhibit or compete with the binding of AXL S6-1, AXL S6-2,and/or AXL S6-5 as provided in Example 2 of the present application. Inyet some other embodiments, sAXL variants of the present inventioninhibit or compete with the binding of any sAXL variant provided inExample 2 of the present application.

The ability of a molecule to bind to GAS6 can be determined, forexample, by the ability of the putative ligand to bind to GAS6 coated onan assay plate. In one embodiment, the binding activity of sAXL variantsof the present invention to a GAS6 can be assayed by either immobilizingthe ligand, e.g., GAS6 or the sAXL variant. For example, the assay caninclude immobilizing GAS6 fused to a His tag onto Ni-activated NTA resinbeads. Agents can be added in an appropriate buffer and the beadsincubated for a period of time at a given temperature. After washes toremove unbound material, the bound protein can be released with, forexample, SDS, buffers with a high pH, and the like and analyzed.

In still yet other embodiments, sAXL variants of the present inventionhas a better thermal stability than the thermal stability of a wild-typeAXL. In some embodiments, the melting temperature of sAXL variants ofthe present invention is at least 5° C., 10° C., 15° C., or 20° C.higher than the melting temperature of a wild-type AXL.

According to the present invention, sAXL variants of the presentinvention can also include one or more modifications that do not alterprimary sequences of the sAXL variants of the present invention. Forexample, such modifications can include chemical derivatization ofpolypeptides, e.g., acetylation, amidation, carboxylation, etc. Suchmodifications can also include modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps; e.g.by exposing the polypeptide to enzymes which affect glycosylation, suchas mammalian glycosylating or deglycosylating enzymes. In someembodiments, sAXL variants of the present invention include sAXL varianthaving phosphorylated amino acid residues, e.g. phosphotyrosine,phosphoserine, or phosphothreonine.

In some other embodiments, sAXL variants of the present inventioninclude sAXL variants further modified to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. For example, sAXLvariants of the present invention further include analogs of a sAXLvariant containing residues other than naturally occurring L-aminoacids, e.g. D-amino acids or non-naturally occurring synthetic aminoacids. D-amino acids may be substituted for some or all of the aminoacid residues.

In yet some other embodiments, sAXL variants of the present inventioninclude at least two same or different sAXL variants linked covalentlyor non-covalently. For example, in some embodiments, sAXL variants ofthe present invention include two, three, four, five, or six same ordifferent sAXL variants linked covalently, e.g., so that they will havethe appropriate size, but avoiding unwanted aggregation.

According to the present invention, sAXL variants of the presentinvention can be produced by any suitable means known or laterdiscovered in the field, e.g., produced from eukaryotic or prokaryoticcells, synthesized in vitro, etc. Where the protein is produced byprokaryotic cells, it may be further processed by unfolding, e.g. heatdenaturation, DTT reduction, etc. and may be further refolded, usingmethods known in the art.

The polypeptides may be prepared by in vitro synthesis, usingconventional methods as known in the art. Various commercial syntheticapparatuses are available, for example, automated synthesizers byApplied Biosystems, Inc., Foster City, Calif., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. Alternatively, RNAcapable of encoding the polypeptides of interest may be chemicallysynthesized. One of skill in the art can readily utilize well-knowncodon usage tables and synthetic methods to provide a suitable codingsequence for any of the polypeptides of the invention. Direct chemicalsynthesis methods include, for example, the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. While chemical synthesis of DNA isoften limited to sequences of about 100 bases, longer sequences can beobtained by the ligation of shorter sequences. Alternatively,subsequences may be cloned and the appropriate subsequences cleavedusing appropriate restriction enzymes.

The nucleic acids may be isolated and obtained in substantial purity.Usually, the nucleic acids, either as DNA or RNA, will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant,” e.g., flanked by one or more nucleotideswith which it is not normally associated on a naturally occurringchromosome. The nucleic acids of the invention can be provided as alinear molecule or within a circular molecule, and can be providedwithin autonomously replicating molecules (vectors) or within moleculeswithout replication sequences. Expression of the nucleic acids can beregulated by their own or by other regulatory sequences known in theart. The nucleic acids of the invention can be introduced into suitablehost cells using a variety of techniques available in the art, such astransferrin polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

In some embodiments, the present invention provides expression vectorsfor in vitro or in vivo expression of one or more sAXL variants of thepresent invention, either consitutively or under one or more regulatoryelements. In some embodiments, the present invention provides a cellpopulation comprising one or more expression vectors for expressing sAXLvariants of the present invention, either consitutively or under one ormore regulatory elements.

According to another aspect of the invention, it provides isolatedantibodies or fragments thereof which specifically binds to a GAS6protein. GAS6 (growth arrest-specific 6) belongs structurally to thefamily of plasma vitamin K-dependent proteins. GAS6 has a highstructural homology with the natural anticoagulant protein S, sharingthe same modular composition and having 40% sequence identity. GAS6 hasgrowth factor-like properties through its interaction with receptortyrosine kinases of the TAM family; Tyro3, AXL and MerTK. Human GAS6 isa 678 amino acid protein that consists of a gamma-carboxyglutamate(Gla)-rich domain that mediates binding to phospholipid membranes, fourepidermal growth factor-like domains, and two laminin G-like (LG)domains. The sequence of the transcript variants of human GAS6 may beaccessed at Genbank at NM_(—)001143946.1; NM_(—)001143945.1; andNM_(—)000820.2, respectively.

GAS6 employs a unique mechanism of action, interacting through itsvitamin K-dependent GLA (gamma-carboxyglutamic acid) module withphosphatidylserine-containing membranes and through its carboxy-terminalLamG domains with the TAM membrane receptors.

According to the present invention, isolated antibodies of the presentinvention include any isolated antibodies with a recognizable bindingspecificity against GAS6. In some embodiments, isolated antibodies arepartially or fully humanized antibodies. In some other embodiments,isolated antibodies are monoclonal or polyclonal antibodies. In yet someother embodiments, isolated antibodies are chimeric antibodies, e.g.,with consistent regions, variable regions and/or CDR3 or a combinationthereof from different sources. In yet some other embodiments, isolatedantibodies are a combination of various features described herein.

According to the present invention, fragments of the isolated antibodiesof the present invention include a polypeptide containing a region ofthe antibody (either in the context of an antibody scaffold or anon-antibody scaffold) that is sufficient or necessary for arecognizable specific binding of the polypeptide towards GAS6. In someembodiments, fragments of the isolated antibodies of the presentinvention include variable light chains, variable heavy chains, one ormore CDRs of heavy chains or light chains or combinations thereof, e.g.,Fab, Fv, etc. In some embodiments, fragments of the isolated antibodiesof the present invention include a polypeptide containing a single chainantibody, e.g., ScFv. In yet some embodiments, fragments of the isolatedantibodies of the present invention include variable regions only orvariable regions in combination with part of Fc region, e.g., CH1region. In still some embodiments, fragments of the isolated antibodiesof the present invention include minibodies, e.g., VL-VH-CH3 ordiabodies.

In some embodiments, isolated antibodies of the present invention bindto an epitope comprised in or presented by one or more amino acidregions that interact with AXL. In some other embodiments, isolatedantibodies of the present invention bind to an epitope comprised in orpresented by one or more amino acid regions of GAS6, e.g., L295-T317,E356-P372, R389-N396, D398-A406, E413-H429, and W450-M468 of GAS6.

In yet some other embodiments, isolated antibodies of the presentinvention bind to an epitope comprised in or presented by one or moreamino acid regions, e.g., LRMFSGTPVIRLRFKRLQPT (SEQ ID NO: 3),EIVGRVTSSGP (SEQ ID NO: 4), RNLVIKVN (SEQ ID NO: 5), DAVMKIAVA (SEQ IDNO: 6), ERGLYHLNLTVGIPFH (SEQ ID NO: 7), and WLNGEDTTIQETVVNRM (SEQ IDNO: 8).

In yet some other embodiments, isolated antibodies of the presentinvention bind to an epitope comprised in or presented by at least one,two, three, four, five, or six amino acids in a region of L295-T317,E356-P372, R389-N396, D398-A406, E413-H429, and W450-M468 of GAS6. Inyet some other embodiments, isolated antibodies of the present inventionbind to an epitope comprised in or presented by at least one, two,three, four, five or six amino acids in a region of LRMFSGTPVIRLRFKRLQPT(SEQ ID NO: 3), EIVGRVTSSGP (SEQ ID NO: 4), RNLVIKVN (SEQ ID NO: 5),DAVMKIAVA (SEQ ID NO: 6), ERGLYHLNLTVGIPFH (SEQ ID NO: 7), andWLNGEDTTIQETVVNRM (SEQ ID NO: 8).

In still some other embodiments, isolated antibodies of the presentinvention is capable of inhibiting, inhibit, or compete with the bindingbetween wild-type AXL or sAXL variants of the present invention andGAS6.

According to the present invention, both sAXL variants and isolatedantibodies of the present invention can be provided in pharmaceuticalcompositions suitable for therapeutic use, e.g., for human treatment. Insome embodiments, pharmaceutical compositions of the present inventioninclude one or more therapeutic entities of the present invention, e.g.,sAXL variants and/or isolated antibodies against GAS6 orpharmaceutically acceptable salts, esters or solvates thereof or anyprodrug thereof. In some other embodiments, pharmaceutical compositionsof the present invention include one or more therapeutic entities of thepresent invention in combination with another cytotoxic agent, e.g.,another anti-tumor agent. In yet some other embodiments, pharmaceuticalcompositions of the present invention include one or more therapeuticentities of the present invention in combination with anotherpharmaceutically acceptable excipient.

In still some other embodiments, therapeutic entities of the presentinvention are often administered as pharmaceutical compositionscomprising an active therapeutic agent, i.e., and a variety of otherpharmaceutically acceptable components. (See Remington's PharmaceuticalScience, 15.sup.th ed., Mack Publishing Company, Easton, Pa., 1980). Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

In still some other embodiments, pharmaceutical compositions of thepresent invention can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

According to yet another aspect of the invention, it provides methodsfor treating, reducing or preventing tumor metastasis or tumor invasionby inhibiting the AXL signaling pathway and/or GAS6 signaling pathway.In some embodiments, methods of the present invention include inhibitingthe activity of AXL, the activity of GAS6, or the interaction betweenAXL and GAS6. For example, the activity of AXL or GAS6 can be inhibitedat the gene expression level, mRNA processing level, translation level,post-translation level, protein activation level, etc. In some otherexamples, the activity of AXL or GAS6 can be inhibited by smallmolecules, biological molecules, e.g., polypeptides, polynucleotides,antibodies, antibody drug conjugates, etc. In some other examples, theactivity of AXL or GAS6 can be inhibited by one or more sAXL variants orisolated antibodies of the present invention.

In yet other embodiments, methods of the present invention includeadministering to a subject in need of treatment a therapeuticallyeffective amount or an effective dose of a therapeutic entity of thepresent invention, e.g., an inhibitor of AXL activity or GAS6 activityor an inhibitor of interaction between AXL and GAS6. In someembodiments, effective doses of the therapeutic entity of the presentinvention, e.g. for the treatment of metastatic cancer, described hereinvary depending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Usually, the patientis a human but nonhuman mammals including transgenic mammals can also betreated. Treatment dosages need to be titrated to optimize safety andefficacy.

In some embodiments, the dosage may range from about 0.0001 to 100mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight orwithin the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per every two weeks or once a month or once every 3to 6 months. Therapeutic entities of the present invention are usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of the therapeutic entity in thepatient. Alternatively, therapeutic entities of the present inventioncan be administered as a sustained release formulation, in which caseless frequent administration is required. Dosage and frequency varydepending on the half-life of the polypeptide in the patient.

In prophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patent canbe administered a prophylactic regime.

In still other embodiments, methods of the present invention includetreating, reducing or preventing tumor metastasis or tumor invasion ofovarian cancer, breast cancer, lung cancer, liver cancer, colon cancer,gallbladder cancer, pancreatic cancer, prostate cancer, and/orglioblastoma.

In still yet some other embodiments, for prophylactic applications,pharmaceutical compositions or medicaments are administered to a patientsusceptible to, or otherwise at risk of a disease or condition in anamount sufficient to eliminate or reduce the risk, lessen the severity,or delay the outset of the disease, including biochemical, histologicand/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presenting during development ofthe disease.

In still yet some other embodiments, for therapeutic applications,therapeutic entities of the present invention are administered to apatient suspected of, or already suffering from such a disease in anamount sufficient to cure, or at least partially arrest, the symptoms ofthe disease (biochemical, histologic and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient response has been achieved. Typically, the response ismonitored and repeated dosages are given if there is a recurrence of thecancer.

According to the present invention, compositions for the treatment ofmetastatic cancer can be administered by parenteral, topical,intravenous, intratumoral, oral, subcutaneous, intraarterial,intracranial, intraperitoneal, intranasal or intramuscular means. Themost typical route of administration is intravenous or intratumoralalthough other routes can be equally effective.

For parenteral administration, compositions of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water, oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998.Co-administration can be achieved by using the components as a mixtureor as linked molecules obtained by chemical crosslinking or expressionas a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin patchor using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24,1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Preferably, a therapeutically effective dose of the antibodycompositions described herein will provide therapeutic benefit withoutcausing substantial toxicity.

Toxicity of the proteins described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The dataobtained from these cell culture assays and animal studies can be usedin formulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics,Ch. 1).

Also within the scope of the invention are kits comprising thecompositions (e.g., soluble AXL variants and formulations thereof) ofthe invention and instructions for use. The kit can further contain aleast one additional reagent. Kits typically include a label indicatingthe intended use of the contents of the kit. The term label includes anywriting, or recorded material supplied on or with the kit, or whichotherwise accompanies the kit.

According to yet another aspect of the invention, it provides methodsfor determining the ability of a tumor to undergo tumor invasion and/ormetastasis by detecting and/or determining the level of AXL activity orGAS6 activity in a biological sample from a subject of interest. In someembodiment, the level of AXL activity or GAS6 activity is measured bythe level of mRNA expression, the level of protein expression, the levelof protein activation or any suitable indicator corresponding to theactivity of AXL or GAS6 either directly or indirectly. In someembodiments, the level of AXL activity or GAS6 activity in a biologicalsample is further compared to a predetermined level, e.g., standardlevel obtained by establishing normal levels or ranges of AXL activityor GAS6 activity based on a population of samples from tumors that donot develop tumor invasion or tumor metastasis or from normal tissues.For example, an increase of AXL activity or GAS6 activity over thepredetermined level or standard level is indicative of a predispositionof the tumor to undergo tumor invasion or tumor metastasis.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible. In thefollowing, examples will be described to illustrate parts of theinvention.

EXPERIMENTAL Example 1 Therapeutic Blockade of AXL Signaling InhibitsMetastatic Tumor Progression

Demonstration of AXL as a therapeutic target for metastatic disease hasbeen largely unexplored, and more importantly no in vivo correlates ofAXL targeting have been demonstrated. We show that AXL is a marker ofmetastases in human breast and ovarian cancer patients, and that theseverity of disease in these patients correlates with the amount of AXLprotein in the primary tumor. Most importantly, we show that tumormetastasis can be successfully treated in mice with pre-existingmetastasis by the administration of soluble AXL ectodomains.Mechanistically, inhibition of AXL signaling in animals with metastaticdisease results in decreased invasion and MMP activity. Our findingsdemonstrate that inhibition of the AXL signaling cascade in tumor cellsthrough the administration of soluble AXL ectodomains is sufficient toinhibit metastatic tumor progression.

In this study, we test whether AXL is a critical factor for metastasisin human cancer and that therapeutic blockade of AXL signaling may be aneffective treatment for metastatic disease. We utilize both genetic andtherapeutic approaches to directly assess the role of AXL in theinitiation and progression of metastatic breast and ovarian cancer.

AXL is a marker of tumor progression and metastases in human cancer. Wefirst compared AXL expression in normal tissue, primary tumor, andmetastases from patients with breast or ovarian cancer. In 100% ofnormal adjacent breast cancer specimens, mammary epithelial cells showeddiffuse cytoplasmic and nuclear staining for AXL that was considered tobe background staining given that AXL is a membrane bound receptor(n=27, FIG. 1A). However in primary breast tumors, membranous AXLstaining in tumor epithelium was present in 25% (¼) of grade 1, 76% (10/13) of grade 2, and 100% (18/18) of grade 3 specimens (FIG. 1A andTable 1). Additionally, AXL was expressed in 88% ( 8/9) of lymph nodemetastases.

In serous ovarian cancer specimens, AXL expression was first examined innormal ovarian surface epithelium (OSE) since the majority of ovariantumors are thought to arise from these cells. In ovarian cancer patientsamples that retained normal OSE, AXL was expressed in 0% (0/5) ofspecimens (FIG. 1B). In contrast, membranous AXL staining in primarytumor epithelium, was present in 66% (6/9) of stage II and 83% ( 53/64)of stage III patient samples (FIG. 1B and Table I). In addition, tumorsamples from common metastatic sites such as the omentum and peritoneumshowed high AXL expression in 75% (24/32) and 90% ( 27/30) of specimensrespectively (FIG. 1B and Table I) These findings demonstrate that AXLexpression within primary tumors correlates with metastasis as shown inadvanced disease and metastatic tumors. Furthermore, these datademonstrate that metastases derived from human breast and ovariancancers express high levels of AXL.

AXL is a critical factor for tumor metastasis. To examine the functionalrole of AXL in metastasis, we utilized a genetic approach to inhibit AXLin mouse models of breast and ovarian metastasis. For this purpose, wescreened a panel of human breast and ovarian cancer cell lines for AXLprotein expression in order to identify metastatic cell lines with highlevels of AXL expression. Similar to our clinical findings, AXL washighly expressed in the majority of metastatic breast (NCI-ADR-RES,MDA-231, HS 578T, 8T-549) and ovarian (SKOV3, OVCAR-8, ES-2, MESOV,HEYA8) cell lines, whereas AXL was expressed at undetectable or lowlevels in cell lines with low metastatic potential (MCF7, MDA-MB435,T47D, IGROV1, OVCAR-3; FIG. 9). AXL deficient metastatic breast(MDA-231) and ovarian (SKOV3ip.1 and OVCAR-8) cell lines were generatedusing previously described AXL shRNA targeting sequences. Western blotanalysis confirmed that cells expressing shAXL targeting sequencesexpressed less than 5% of AXL protein compared to cells expressing thescramble control shRNA targeting sequence (shSCRM, FIG. 9B).

To directly assess the role of AXL in the late stages of breast tumormetastasis, we injected AXL-wildtype (shSCRM) and AXL-deficient (shAXL)MDA-231 cells into the tail vein of nude mice and evaluated tumor burdenin the lungs at day twenty-eight. Microscopic evaluation of lungsrevealed that 5/5 mice injected with shRNA scramble (shSCRM) MDA-231cells developed metastatic foci that stained positive for AXL (FIG. 2A).In contrast, 0/5 mice injected shRNA AXL (shAXL) developed lungmetastases upon histologic evaluation (FIG. 2A). In order to quantifytumor burden in the lungs of these mice, we performed real time PCRanalysis for human GAPDH. FIG. 2A demonstrates that the lungs of miceinjected with shSCRM MDA-231 cells expressed high levels of human GAPDHindicating the presence of metastatic lesions derived from MDA-231cells. In addition, shSCRM injected mice expressed human AXL in the lungsuggesting the presence of AXL positive tumor cells (FIG. 2A). Incontrast, mice injected with shAXL tumor cells did not express humanGAPDH or AXL in the lung. These findings demonstrate that geneticinactivation of AXL is sufficient to completely suppress the formationof lung metastasis in this model.

To determine whether genetic inactivation of AXL affects the ability ofovarian cancer cells to metastasize in vivo, we compared the ability ofshSCRM and shAXL SKOV3ip.1 cells to form metastases using a peritonealxenograft model of ovarian cancer. This model recapitulates theperitoneal dissemination of human ovarian metastases in which micedevelop rapidly progressive disease consisting of ascites and more than100 small metastatic lesions attached to the mesentery, diaphragm,liver, and other peritoneal surfaces following peritoneal injection ofSKOV3ip.1 cells (FIG. 3B). Immunohistochemical analysis of AXLexpression in SKOV3ip.1 peritoneal metastases revealed that similar tohuman ovarian metastases, AXL is highly expressed in SKOV3ip.1metastatic lesions, indicating that this is a relevant model system toinvestigate the role of AXL in ovarian metastasis (data not shown).Twenty-eight days following peritoneal injection of shSCRM and shAXLcells, shSCRM mice displayed signs of severe ascites and morbiditynecessitating us to sacrifice the mice and investigate changes in tumorburden between the shSCRM and shAXL injected mice. While mice injectedwith shSCRM cells developed ascites and >100 peritoneal metastases, miceinjected with shAXL cells developed very few metastases (FIG. 2B). Theaverage number of peritoneal metastases greater than 5 mm in size wassignificantly reduced from 13.4+/−4.3 in shSCRM injected mice to0.8+/−0.5 in shAXL injected mice (FIG. 2B). Similarly, the averageweight of these tumors was significantly reduced from 236+/−74 mg inshSCRM-injected mice to 39.2+/−18 mg in shAXL-injected mice (FIG. 2B).In support of these findings, knockdown of AXL expression in OVCAR-8cells significantly inhibited total ovarian peritoneal tumor mass andtumor number (FIG. 2C). Collectively, these findings demonstrate thatAXL is a critical factor for breast and ovarian tumor metastasis.

Given the important role of AXL in the formation metastasis in vivo, wenext sought to determine if AXL specifically regulates metastasis, or ifAXL plays a general role in the regulation of tumor cell proliferationand growth. To address these questions, we performed in vitroproliferation assays in which total cell numbers between AXL wild type(shSCRM) and AXL deficient (shAXL) cells were counted over a 10-14 dayperiod. We found no significant difference in cellular growth curvesbetween shSCRM and shAXL MDA-231, SKOV3ip.1 or OVCAR-8 cells (FIG. 3).Similarly, no significant difference was observed orthotopic MDA-231 orsubcutaneous SKOV3ip.1 tumor growth between shSCRM and shAXL cells (FIG.3). These findings indicate that AXL is not required tumor cellproliferation or subcutaneous growth in vivo. Overall, our findingsindicate that AXL specifically regulates tumor metastasis in breast andovarian tumors.

AXL regulates tumor cell invasion. To determine a potential mechanismfor AXL-mediated metastasis, we took an unbiased approach and directlycompared the role of AXL in the critical cellular functions associatedwith the metastatic cascade including proliferation, invasion,migration, adhesion, and survival}. We found that shAXL MDA-231,SKOV3ip.1, and OVCAR-8 cells were significantly impaired in the abilityto invade through type I collagen (FIG. 4A). We also observed a modestdecrease in cellular migration in shAXL cells, yet we were unable tofind a difference in adhesion to ECM proteins or survival followingserum withdrawal indicating that AXL predominately affects invasion inthe metastatic cascade.

At the molecular level, MMP-9 has recently been identified as aneffecter of AXL-mediated invasion in breast cancer cells. Therefore, weinvestigated whether MMP-9 expression or activity was also altered inAXL-deficient ovarian tumor cells. While SKOV3ip.1 cells do not expressMMP-9, we found that MMP-2 was highly expressed in these cells and MMP-2mRNA was significantly decreased in shAXL cells (FIG. 4B). MMP-2luciferase reporter assays revealed that MMP-2 promoter activity wassignificantly decreased in shAXL cells compared to shSCRM cellsindicating that AXL regulates MMP-2 at the transcriptional level (FIG.4C). Gelatin zymography assays indicated that MMP-2 secreted proteinlevels were also significantly reduced in shAXL cells compared to shSCRMSKOV3ip.1 cells (FIG. 4D). Collectively, these findings suggest a rolefor AXL as an upstream regulator of MMP-2 expression and activity inhuman ovarian cancer cells.

We next sought to elucidate the signaling pathways involved inAXL-mediated MMP-2 expression. Activation of AXL by GAS6 has beenreported to directly induce a number of intracellular signaling pathwaysincluding PI3K, RAS, MAPK, SRC, and PLC. Among these pathways, the PI3Ksignaling pathway has been shown to regulate MMP-2 expression andinvasion in ovarian cancer cells. To determine whether PI3K signaling isaffected by loss of AXL in SKOV3ip.1 cells, we performed western blotanalysis for phospho-AKT at Ser473 (P-AKT) in AXL-wild type andAXL-deficient SKOV3ip.1 cells. We found a profound inhibition of P-AKTexpression in shAXL cells compared to shSCRM SKOV3ip.1 cells (FIG. 4E).Additionally, GAS6 stimulation of starved SKOV3ip.1 cells resulted in aPI3K-dependent induction of P-AKT as treatment with the PI3K inhibitorLy294002 completely abrogated GAS6-induced P-AKT expression (FIG. 4E).To determine whether the PI3K pathway was involved in AXL-mediated MMP-2expression, we performed MMP-2 luciferase reporter assays in thepresence of GAS6 and Ly294002. The induction of MMP-2 promoter activityfollowing GAS6 stimulation was completely blocked by Ly294002 treatmentsuggesting that GAS6/AXL signaling regulates MMP-2 expression throughthe PI3K signaling events (FIG. 4F).

Therapeutic inhibition of AXL significantly suppresses metastatic tumorprogression in mice. Our findings thus far demonstrate that AXL is acritical factor for metastasis and support the hypothesis thattherapeutic blockade may be an effective treatment for metastaticdisease. To test this hypothesis, we utilized the soluble AXL ectodomainas a therapeutic strategy to inhibit AXL signaling. The soluble AXLectodomain functionally acts as a decoy receptor and has previously beenshown to bind GAS6 with nanomolar affinity in vitro and in vivo (FIG.5A). We first examined whether treatment with soluble AXL ectodomains issufficient to inhibit AXL signaling and invasion in metastatic tumorcells. PI3K/AKT signaling is regulated by AXL in a variety of celltypes. We found that PI3K/AKT signaling is regulated by GAS6/AXLsignaling in SKOV3ip.1 cells and treatment with soluble AXL ectodomains(sAXL) was able to reduce PI3K/AKT activation in GAS6 treated SKOV3ip.1cells (FIGS. 5B and C). Similarly, treatment of MDA-231 cells incollagen with sAXL was sufficient to dramatically reduce cellularinvasion demonstrating that sAXL treatment affects AXL signaling andinvasion in vitro (FIG. 5D).

We next examined whether sAXL treatment would affect metastatic tumorprogression in the highly metastatic models of ovarian cancer. We firstestablished SKOV3ip.1 metastatic lesions in nude mice (day 1) and begantreatment with sAXL at day 7 following verification of macroscopiclesions. sAXL therapy was delivered using the adenoviral system in whichthe liver releases systemic production of sAXL protein into the serum ofmice for up to 28 days following injection (FIG. 6A). Macroscopicanalysis of tumor burden at day 28 revealed that mice receiving sAXLtherapy had a significant (p<0.01) reduction in tumor burden compared tomice treated with the Fc control therapy. In the SKOV3ip.1 tumor model,total tumor weight and tumor number was decreased by 63% in mice treatedwith sAXL compared to Fc treated mice (FIG. 6B). Similarly in theOVCAR-8 model, total tumor weight and tumor number was significantlydecreased by 47% and 42% respectively (FIG. 11). We examined MMP2expression levels in SKOV3ip.1 tumors by real time PCR analysis andfound that MMP2 levels were significantly decreased in the tumors ofsAXL treated mice compared to Fc control treated mice (FIG. 6C). Theseresults demonstrate that single agent AXL therapy is sufficient tosignificantly reduce metastatic tumor burden in mice with establisheddisease. In addition, our findings suggest that the therapeutic effectof AXL on metastatic tumor growth may involve the inhibition of invasionat least in part through the regulation of MMP activity.

Given that previous anti-metastatic inhibitors that target MMPs havebeen shown to have significant effects on normal tissue toxicity, weperformed a comprehensive analysis of normal tissue toxicity in micetreated with sAXL therapy for 21 days. We observed no behavioral,macroscopic, or microscopic abnormalities in nude mice treated with sAXLor Fc therapy (FIG. 7).

Invasion and migration are important cell intrinsic properties thatcontribute to the pathogenesis of tumor metastasis. It has beenhypothesized that therapeutic agents targeting these processes may be auseful strategy to inhibit metastasis and may provide clinical benefitsto patients with metastatic disease. In this report, we demonstrate thatthe receptor tyrosine kinase AXL is a critical factor governing tumorcell invasion and metastasis. Most importantly, we show that therapeuticblockade of AXL signaling using soluble AXL receptors is sufficient tosignificantly inhibit metastatic tumor progression in mice withpre-existing metastatic disease. Mechanistically, our studies indicatethat soluble AXL therapy inhibits tumor metastasis at least in partthrough the inhibition of MMP activity and invasion. Finally, we showthat AXL is highly expressed in metastases and advanced stage primarytumors from human ovarian and breast cancer patients highlighting theclinical importance of our findings.

It is demonstrated herein that AXL is a critical factor for metastasisin human cancer and that therapeutic blockade of AXL signaling is aneffective treatment for metastatic disease. Here we demonstrate that AXLis highly expressed in metastases and advanced primary tumors samplesfrom breast and ovarian cancer patients. We demonstrate genetically thatAXL is critical for the initiation of metastatic breast and ovariancancer using disease using nude mouse models. Most importantly, we havedeveloped highly specific and non-toxic soluble AXL receptors as ananti-AXL therapy and demonstrate that soluble AXL receptor therapy issufficient to significantly inhibit metastatic tumor progression in micewith pre-existing metastatic disease. Our findings demonstrate thatinhibition of the AXL signaling cascade in tumor cells can block boththe initiation and progression of metastatic disease. Our data implicateAXL as a new therapeutic target for advanced and metastatic breast andovarian cancer and suggest that anti-AXL therapy may control both theinitiation and progression of metastatic disease.

MMPs play an important role in the regulation of tumor cell invasion andmetastasis.

However, the mechanisms by which tumor cells induce MMP activity remainunclear. MMP expression is increased in human cancer and correlates withtumor progression and poor patient survival. Gene amplifications andactivating mutations in MMPs are rarely found in human cancer suggestingthat other factors are responsible for enhanced MMP expression incancer. Our data provide evidence that MMP-2 expression is regulated byAXL at the transcriptional level in human ovarian cancer cells. Whilethe exact mechanisms by which AXL regulates MMP-2 expression remain tobe determined, we demonstrate that pharmacological inhibition of thePI3K pathway reduces MMP-2 promoter activity in GAS6 stimulated cellsindicating a role for the PI3K pathway (FIG. 8). Importantly, ourresults indicate that therapeutic blockade of AXL may be an effectiveand non-toxic strategy to inhibit MMP activity in tumors. Broad-spectrumMMP inhibitors were unsuccessful in cancer trials in part due to highlevels of normal tissue toxicity. Our findings indicate that predictedside effects of anti-AXL therapy are minimal. We did not observe anynormal tissue toxicity associated with adenoviral-mediated delivery ofsoluble AXL ectodomain therapy in mice. Furthermore, germline AXL andGAS6 knockout mice are viable and phenotypically normal as adultssuggesting that AXL or GAS6 are not required for development or normaltissue function.

We show that single-agent AXL therapy is sufficient to inhibitmetastatic tumor progression in highly metastatic models of metastaticovarian cancer. These findings have important clinical implications forthe treatment of ovarian cancer. Approximately 14,600 people die fromovarian cancer each year in the United States. Currently there are noFDA approved biologics for the treatment of ovarian cancer, althoughAvastin (mAb targeting VEGF) and Tarceva (small molecule EGFR kinaseinhibitor) are in clinical trials for the treatment of advanced andrecurrent ovarian cancer. Standard therapy for ovarian cancer includessurgery with optimal debulking of disease followed by cytotoxicplatinum-taxane combination therapy. Despite these efforts, eightypercent of patients diagnosed with ovarian cancer develop recurrentdisease and only 30% of these patients survive 5 years followingdiagnosis.

Our data show that AXL therapy is an effective adjuvant therapy for thetreatment of advanced and recurrent ovarian cancer. The model ofmetastatic ovarian tumor progression used in our studies resembles thedevelopment of recurrent disease in human patients following surgicaldebulking. We found that AXL therapy was able to reduce metastatic tumorburden in mice with established disease by 63%. The establishment of newmetastatic lesions during the progression of disease was significantlyreduced. This observation is consistent with our findings demonstratingthat AXL predominantly affects tumor cell invasion rather than cellularproliferation or growth. Taken together our results indicate that AXLtherapy functions primarily as an anti-metastatic agent and may be mosteffective as a combination therapy with current cytotoxic agents.

In summary, AXL is a critical factor for metastasis and blockade of AXLsignaling has therapeutic benefits in metastasis. These studies provideimportant pre-clinical data for anti-AXL therapy for metastatic disease.

Methods

Cell Lines. Ovarian SKOV3, SKOV3ip.1, and HEYA8 cells were obtained as agift from Dr. Gordon Mills (MD Anderson Cancer Center). Ovarian ES-2 andMESOV cells were a gift from Dr. Branimir Sikic (Stanford University).MDA-231, OVCAR-3, and MCF-7 cells were purchased from ATCC. IGROV-1 andOVCAR-8 cells were purchased from the NCI-Frederick DCTD tumor cell linerepository. Cells were cultured in the appropriate media supplementedwith 10% heat inactivated fetal bovine serum and 1% penicillin andstreptomycin at 37° C. in a 5% CO₂ incubator. Cell pellets from theNCI60 panel of breast and ovarian cancer cell lines were provided by Dr.Giovani Melillo (NCI-Frederick).

Patients and Tissue Microarrays. Human breast tissue microarrays werepurchased from US Biomax (BR1002). Ovarian human tissue microarrays wereobtained from the Stanford University Pathology Department. A total of73 paraffin embedded tumor samples were obtained from previouslyuntreated ovarian cancer patients at Stanford Hospital from 1995 to2001. These primary ovarian tumor samples were assembled into a tissuemicroarray consisting of two samples per patient. An additional 30 tumorsamples from the peritoneum were also evaluated in this microarray. Allpatients had serous ovarian cancer, and staging information was obtainedaccording to the International Federation of Gynecology and Obstetricsstandards. All specimens and their corresponding clinical informationwere collected under protocols approved by the institutional reviewboard at Stanford University. An additional commercially available tumormicroarray was used to examine 32 metastatic lesions from the omentum(US Biomax).

AXL Immunohistochemistry. Paraffin embedded tissue slides weredeparaffinized with xylene, rehydrated, and unmasked following standardimmunohistochemical methods. The AXL primary antibody (RandD Systems)was used at a 1:500 dilution. Negative controls for all samples weredone using the secondary antibody alone. Antigen-antibody complexes werevisualized using the VECTASTAIN ABC system (Vector Laboratories) and DABSubstrate Kit for Peroxidase (Vector Laboratories) following theprotocols of the manufacturer. Slides were counterstained withhematoxylin. AXL staining on the membrane of tumor cells was scoredmicroscopically according to the percentage of cells positive for AXLexpression (0 for absence, 1 for poor quality sample, 2 for 5-60%, and 3for 61-100%).

Reporter Assays. The MMP-2 reporter plasmid driven by 1659 by of theMMP-2 promoter was a gift. Luciferase activity was determined byDual-Glo Luciferase Assay reagent (Promega) in shSCRM and shAXLSKOV3ip.1 cells and measured in a Monolight 2010 Luminometer (AnalyticalLuminescence Laboratory). Firefly luciferase activity was normalized toRenilla activity. Assays were performed in triplicate and were repeatedtwice.

Transient and Retroviral Transfections. Transient DNA transfections wereperformed with Lipofectamine 2000 (Invitrogen) in accordance with themanufacturers instructions. 0.1 μg of MMP-2 cDNA (OpenBiosystems) wastransfected into a 6 well dish.

siRNA: siRNA sequences targeting AXL or control were purchased formDharmacon. All siRNA transfections were carried out using DharmaconSmart Pools with Dharmafect 1 transfection reagent according tomanufacturer's protocol (Dharmacon, Lafayette, Colo.).

shRNA: Oligos for the specific degradation of AXL RNA were synthesizedas previously described [SEQ ID NO:8] 5′-GATTTGGAGAACACACTGA-3′. Ascramble sequence was used as a non-targeting shRNA [SEQ ID NO:9]5′-AATTGTACTACACAAAAGTAC-3′. These oligos were cloned into theRNAi-Ready pSiren RetroQ (BD Bioscience) vector and SKOV3ip.1, MDA-231,and OVCAR-8 cells were retrovirally transduced with these vectors.Infected cells were selected in puromycin (Sigma) and polyclonalpopulations were tested for decrease AXL expression levels by westernblot analysis.

Plasmids. The AXL ectodomain corresponding to amino acids 1-451 wasamplified from the human AXL cDNA (Open Biosystems) and cloned into theCMV-driven pADD2 adenoviral shuttle vector. Transient DNA transfectionswith control vector or AXL 1-451 were performed with Lipofectamine 2000(Invitrogen) in accordance with the manufacturers into HCT116 cells.Conditioned media was collected 48-72 hours following transfection.

Adhesion Assays. SKOV3ip.1 shSCRM and shAXL cells were fluorescentlylabeled with 5 um CMFDA (Molecular Probes). Cells were washed anddetached using a non-enzymatic cell dissociation buffer (Gibco). Cells(5×10e5) were plated into a 96 well plate and precoated with 50 ug/ul ofcollagen type I (BD Bioscience). After a 60-minute incubation at 37 C,cells were carefully washed 5 times. Fluorescent activity (excitation,494 nm; emission, 517 nm) was measured using a fluorescentspectrophotometer.

SKOV3ip.1 Adhesion to Collagen Typel. SKOV3ip.1 shSCRM and shAXL cellswere fluorescently labeled with 5 um CMFDA (Molecular Probes). Cellswere washed and detached using a non-enzymatic cell dissociation buffer(Gibco). Cells (5×10e5) were plated in triplicate into a 96 well plateand precoated with 50 ug/ul of collagen type I (BD Bioscience). After a60-minute incubation at 37 C, cells were carefully washed 5 times.Fluorescent activity (excitation, 494 nm; emission, 517 nm) was measuredusing a fluorescent spectrophotometer.

MDA-231 Adhesion to ECM Proteins. MDA-231 shSCRM and shAXL (0.5×10^6)cells were plated in triplicate onto wells containing an array of ECMproteins including laminin, collagen I and IV, fibronectin, andfibrinogen. Cells were incubated at 37 C for 1 hr and washed in PBS.Adherent cells were stained and quantified at OD 560 according to themanufacturer's protocol (CellBiolabs).

Migration Assays. Cellular migration was examined in vitro as previouslydescribed. Briefly, cells were serum-deprived for 24 hr and seeded(2.5×10⁴ cells) in triplicate onto uncoated inserts (BD Biosciences),moved to chambers containing FBS as chemo-attractant and incubated for24 hr. After removing the non-invading cells, the cells at the bottomside of the membranes were fixed, stained and counted. Five fields werecounted for each membrane. The % migration was determined as follows:(average # of cells migrating in shAXL cells/average # of cellsmigrating in shSCRM cells)×100. Experiments were performed in triplicateand repeated three times.

Collagen Invasion Assay. Collagen invasion assays were performed aspreviously described. Briefly, 533 cells were plated into collagen typeI on a 48 well plate. Cells were cultured in standard media or mediawith the addition of conditioned control media or sAXL-conditioned mediafor 5-7 days and photographs were taken. Invasion through collagen wasquantified by calculating the percentage of tumor cells that displayed abranching phenotype per 20× field. Three fields per sample were counted.Experiments were performed in triplicate and repeated 2 times.

Gelatin Substrate Zymography. SKOV3ip.1 shSCRM and shAXL cells wereserum starved for 48 hours. 25,000 cells were plated into a 96 wellplate and conditioned media was collected 24 hours later. Equal volumesof conditioned media were run under non-reducing conditions on 10%zymogram gels (Invitrogen). After electrophoresis, gels were washed in2.5% (v/v) Triton X-100 to remove SDS and washed in 50 mM Tris-HCl, 5 mMCaCl₂, and 0.1% Triton X-100 (pH 7.8) and incubated overnight at 37° C.Zymograms were stained for 30 min with 0.25% (w/v) Coomassie BrilliantBlue R250 dissolved in 40% methanol and 10% glacial acetic acid. Gelswere distained in 40% methanol and 10% glacial acetic acid. Experimentswere performed in duplicate and repeated three times.

Cell Proliferation Assays. For monolayer growth curves, cells (50,000)were plated into 60 mm dishes in triplicate. Every three days, the cellswere trypsinized, counted using a cell counter (coulter counter) and50,000 cells were replated and counted.

XTT Survival Assay. Cell viability was measured by the XTT assay aspreviously described. Briefly, serum fed or starved cells (0, 3, 6, and7 days) were incubated with phenol red-free medium with 0.3 mg/mL XTTand 2.65 μg/mL N-methyl dibenzopyrazine methyl sulfate. The 96-wellplates were returned to the 37° C. incubator for 1 to 2 h. Metabolism ofXTT was quantified by measuring the absorbance at 450 nm.

Protein Isolation and Western Blot Analysis. Protein lysates wereharvested in 9M Urea, 0.075M Tris buffer (pH 7.6). Protein lysates werequantified using the Bradford assay, and subjected to reducing SDS-PAGEusing standard methods. Western blots were probed with antibodiesagainst AXL (RandD Systems), alpha Tubulin (Fitzgerald Antibodies), AKT(Cell Signaling), phospho-AKT (Cell Signaling).

For GAS6 stimulation, cells were serum starved for 24 hours. Cells werethen treated with 25 um of PI3K inhibitor (Ly294002, Bio Mol ResearchLaboratory) or 100 l of conditioned media containing the AXL Ecto domainfor 4 hours before treatment with 400 ng/ml of GAS6 for 15 minutes.

For analysis of sAXL expression in the serum of mice, 1.5 l of serumfrom each samples was analyzed by gel electrophoresis.

Generation and Production of Adenovirus. The AXL ectodomaincorresponding to amino acids 1-451 was amplified from the AXL cDNA (OpenBiosystems) and cloned into the E1 region of E1⁻E3⁻ Ad strain 5 byhomologous recombination followed by adenovirus production in 293 cellsand CsCl gradient purification as previously described. The productionand purification of the sAXL adenovirus was performed as previouslydescribed. The generation and production of the negative control virusexpressing murine IgG2-Fc immunoglobulin fragment has been previouslydescribed.

Growth of SKOV3ip.1 and OVCAR-8 Cells as Peritoneal Xenografts. Allprocedures involving animals and their care were approved by theInstitutional Animal Care and Usage Committee of Stanford University inaccordance with institutional and NIH guidelines.

Control and AXL SKOV3ip.1 and OVCAR-8 cells were injected i.p. with1×10⁶ and 5×10⁶ cells respectively in 0.5 ml of PBS into female nudemice. After sacrifice, ascites was quantified, metastatic lesions werecounted, and all visible lesions were dissected and removed to weightumor weight.

SKOV3ip.1 and OVCAR-8 parental cells were injected i.p. with 1×10⁶ and5×10⁶ cells respectively in 0.5 ml of PBS into female nude mice. Seven(SKOV3ip.1) or 14 (OVCAR-8) days following tumor cell injection, micewere injected with sAXL or control 1.9×10⁸ adenoviral pfu in 0.1 ml PBSinto the tail vein. After sacrifice, ascites was quantified, metastaticlesions were counted, and all visible lesions were dissected and removedto weigh total tumor weight.

Tissue Toxicity Studies. SKOV3ip.1 parental cells were injected i.p.with 1×10⁶ and 5×10⁸ cells respectively in 0.5 ml of PBS into femalenude mice. Seven days following tumor cell injection, mice were injectedwith sAXL or control 1.9×10⁸ adenoviral pfu in 0.1 ml PBS into the tailvein. At day 28, mice were sacrificed. Blood was collected and acomprehensive metabolic panel and CBC analysis was performed by theDepartment of Comparative Medicine at Stanford University. Tissuesamples were collected from all major organs including liver, kidney,brain, and spleen, fixed in 10% formalin, embedded in paraffin,sectioned, and counter stained with hematoxylin and eosin.

In vivo Tail-Vein Metastasis Assay. Control and AXL shRNA MDA-231 cellswere injected intravenously with 5×10⁵ cells in 0.1 ml of PBS into thetail vein of nude mice. Four weeks after injection, mice weresacrificed. Microscopic evaluation of lung foci was performed onrepresentative cross-sections of formalin-fixed, paraffin-embedded lungsstained with haematoxylin and eosin. The correct identification of lungfoci with a minimum of four human cells with large nuclei and positivefor AXL expression was confirmed by a board-certified pathologist. Tumorburden in the lungs of mice was quantified by real time PCR analysis ofhuman GAPDH and AXL expression in RNA isolated from whole lung.

Growth of MDA-231 Cells as Orthotopic Tumors. MDA-231 cells were grownas subcutaneous orthotopic tumors in six-week-old female Nude (nu/nu)mice after intradermal injection of 10⁷ cells in 0.1 ml of PBS into themammary fat pad. Tumors were measured with calipers over a 38-day timecourse. Volume was calculated using the following formula:width²×length×0.5.

Growth of SKOV3ip.1 Cells as Subcutaneous Tumors. Five million cells in0.1 ml of PBS were implanted subcutaneously into the flanks Nude (nu/nu)six-week-old female mice. Tumors were measured with calipers over a45-day time course. Volume was calculated using the following formula:width² X length×0.5.

RNA and Real Time PCR Analysis. RNA was isolated from cells and tissuesusing trizol according to manufacturer's protocols (Invitrogen). cDNAwas synthesized from 2 μg of DNase (Invitrogen)-treated RNA using theSuperScript first-strand synthesis system for reverse transcription-PCR(Invitrogen). One microliter of cDNA was subjected to PCR amplificationusing SYBR GREEN PCR Master Mix (Applied Biosystems). The followingprimer sets were used to amplify specific target genes: 18S FWD: [SEQ IDNO:10] 5-GCCCGAAGCGTTTACTTTGA-3 REV: [SEQ ID NO:11]5-TCCATTATTCCTAGCTGCGGTATC-3; AXL FWD: [SEQ ID NO:12]5-GTGGGCAACCCAGGGAATATC-3 REV: [SEQ ID NO:13] 5-GTACTGTCCCGTGTCGGAAAG;GAPDH [SEQ ID NO:14] 5-ATGGGGAAGGTGAAGGTCG-3 REV: [SEQ ID NO:15]5-GGGGTCATTGATGGCAACAATA-3; MMP-2 FWD: [SEQ ID NO:16]5-GCCCCAGACAGGTGATCTTG-3 REV [SEQ ID NO:17] 5-GCTTGCGAGGGAAGAAGTTGT-3.PCR amplification was performed on the Prism 7900 Sequence DetectionSystem (Applied Biosystems). The thermal-cycling profile used wasdenaturation at 50° C. for 2 min and 95° C. for 10 min, followed bycycles of denaturation at 95° C. for 15 s and 60° C. for 1 min. 18S wasused to normalize mRNA. Relative mRNA expression levels were determinedusing the relative standard curve method according to the manufacturersinstructions (Applied Biosystems).

Statistical Analysis. Tests for an association between AXL expressionand tumor formation and metastasis was performed using the Fisher'sexact test. All other statistical tests were performed using theStudent's t test. Values with a p value of <0.05 were consideredstatistically significant.

Abbreviations: GAS6, growth arrest specific gene 6; MMP-2, matrixmetalloproteinase; EOC, epithelial ovarian cancer; ECM, extracellularmatrix; AKT, v-akt murine thymoma viral oncogene homolog.

TABLE 1 Statistical Analysis of AXL Staining to Tumor Parameters Score 01 2 3 Total Breast Infiltrating ductal carcinoma Grade 1 3 (75) 0 (0)  0(0)  1 (25) 4 Grade 2 3 (23) 0 (0)  5 (38)  5 (38) 13 Grade 3 0 (0) 0(0)  7 (39) 11 (61) 18 Totals 6 0 12 17 35 Pearson X2 P value =Metasatic Infiltrating ductal carcinoma Lymph node 0 (0) 1 (11)  4 (44) 4 (44) 9 Ovarian Serous adenocarcinoma Stage II 3 (33) 0 (0)  3 (33)  3(33) 9 Stage III/IV 6 (9) 5 (8) 14 (22) 39 (61) 64 Totals 9 5 17 42 73Pearson X2 P value = Metasatic serous adenocarcinoma Omentum 3 (9) 5(16)  6 (19) 18 (56) 32 Peritoneum 1 (3) 2 (7) 12 (40) 15 (50) 30 Totals4 7 18 33 62 Values are represented as n (%). For tumor cells,membranous staining was scored as 0, absence; 1, unable to score; 2, 5to 60% positive; 3, 61 to 100% positive.

Example 2

We showed that inhibition of the GAS6 ligand binding to cellular AXLthrough overexpression of wild-type soluble AXL in mice using anadenoviral expression system resulted in decreased tumor burden, asmeasured by tumor number and size, compared to untreated control,further highlighting the importance of GAS6 and AXL as critical targetsand effective strategies to inhibit the progression of metastasis inpre-clinical mouse models.

Engineered soluble variants of the AXL extracellular domain are providedherein, which have high affinity for the ligand GAS6, allowing them tosequester the ligand and diminish endogenous AXL signaling. Engineeredvariants have substantially improved affinity for Gas6 compared towild-type AXL.

The extracellular domain of AXL comprises two IgG-like domains and twofibronectin-like domains. The major GAS6 binding site is in the Ig1domain, and the minor GAS6 binding site is in the Ig2 domain.

To further enhance the affinity of the major binding site, we engineeredthe Ig1 domain with break points of 19-132 corresponding to the AXLSwissProt entry P30530. A mutant library was created by performingerror-prone PCR on the Ig1 domain of the AXL receptor using standardmolecular biology techniques. The library was expressed using yeastsurface display and screened by fluorescence-activated cell sorting(FACS) to isolate mutants which exhibit enhanced binding affinity tosoluble GAS6. In our library screening approach, the mutant proteinlibrary was subjected to multiple rounds of sorting wherein eachsuccessive round reduces the size of the library while concurrentlyenriching for desired mutant protein property, which in this case ishigh affinity binding to GAS6.

In order to obtain AXL mutants with significantly strong affinity forGAS6, in the later sorting rounds we used “off-rate” sorts. For off-ratesorts, the library of protein mutants is first incubated with solubleGAS6 and then washed with buffer to remove unbound GAS6 from thesolution. Next, the mutant library is incubated in the presence ofexcess soluble competitor for 2, 4, 6, 12, or 24 hr at room temperature.The excess competitor serves to sequester GAS6 that dissociates fromyeast-displayed AXL, rendering the unbinding step irreversible. MutantAXL proteins that retain binding to GAS6 are collected using FACS.Analysis of the binding to GAS6 by the pooled sort 5 products followingoff-rate steps of 0, 4 and 6 hours shows these products exhibitsignificant improvement over wild-type AXL in terms of persistentbinding to GAS6 (see FIG. 12). The bar graph quantifies the data fromthe dot plots, demonstrating significant improvement of the librarymembers. Sequencing of these products identified several mutationswithin the Axl Ig1 domain that confer the enhanced affinity towards Gas6observed for the pooled sort 5 products (FIG. 12 and Table 2). A 6^(th)round of sorting further enriched to 3 specific clones from the sort 5products. Table 2 shows unique amino acid mutations within the AXLsequence that are contained in the sort round 5 and round 6 products. Inthis table, the residue number in the top row indicates the amino acidresidue in wild-type AXL. The second row indicates the residue found inwild-type AXL at the given position. In subsequent rows, amino acidmutations present in the given mutant are specified. Absence of an aminoacid for a particular residue within a mutant (e.g. a blank space or ablank cell in Table 2) denotes that this amino acid residue is notmutated from the wild-type residue. The standard single letterdesignation for amino acid residues is used as is well-understood by onewho is skilled in the art.

Shown are unique sequences from the sort 5 and 6 products, as well asthe binding properties of the pooled clones as compared to wild-typeAXL, demonstrating substantial improvement in GAS6 binding for thepooled sort 5 products.

Mutants isolated using this directed evolution approach include theamino acid substitutions shown in Table 3.

TABLE 3 Mutants Isolated Using Directed Evolution 26 32 33 74 79 87 92127 Wt-AXL E G N S V D V G Axl S6-1 S G A R Axl S6-2 G M A E Axl S6-5 SN G A

According to the crystal structure of the GAS6-AXL complex reported bySasaki et al. (EMBO J 2006), all mutations shown above, except for E26G,G32S, N33S and G127R/E, lie at the binding interface between AXL andGAS6.

Individual mutants, AXL S6-1 and AXL S6-2, from the sixth round ofsorting were selected for further investigation. Equilibrium bindingtitrations of wild-type AXL, AXL S6-1, and AXL S6-2 were conducted tocompare affinity of the interaction with GAS6 of the wild-type or mutantAXL proteins. The data was fit to a four-point sigmoidal curve and themidpoint was taken as the equilibrium binding constant, K_(D). Themutants AXL S6-1 and AXL S6-2 exhibit substantial improvements in GAS6binding affinity compared to wild-type AXL (FIG. 13 and Table 4).Wild-type AXL has a binding affinity (K_(D)) towards Gas6 of2.4±1.2×10⁻⁹ M; AXL S6-2 has a binding affinity (K_(D)) towards Gas6 of1.89±0.37×10⁻¹⁰ M towards Gas6; and AXL S6-1 has a binding affinity(K_(D)) of 1.12±0.23×10⁻¹⁰ M towards Gas6. For AXL S6-1 and AXL S6-2,this is a 22-fold and 12.8-fold stronger GAS6 binding affinity,respectively, compared to wild-type AXL (Table 4).

TABLE 4 Binding affinity (K_(D)) of wild-type and mutant AXL proteins toGas6. Equilibrium Gas6 Binding K_(D) (M) +/− (M) fold over wt wt AXL 2.4 × 10⁻⁹  1.2 × 10⁻⁹ — S6-1 1.12 × 10¹⁰ 0.23 × 10⁻¹⁰ 22 S6-2 1.89 ×10⁻¹⁰ 0.37 × 10⁻¹⁰ 12.8

We also investigated the thermal stability of wild-type and mutant AXLproteins using variable temperature circular dichroism scans. Thistechnique monitors the unfolding of the secondary structural elements ofthe folded protein as a function of temperature. Ellipticity of eachprotein was monitored as a function of temperature and the data was fitto a standard two-state unfolding curve. The melting temperature (T_(m))is the midpoint of the unfolding curve. Wild-type AXL exhibited amelting temperature of 41.3±0.6° C.; AXL S6-1 exhibited a meltingtemperature of 54.0±0.9° C. (approximately 13° C. higher thermalstability than wild-type AXL); Axl S6-2 exhibited a melting temperatureof 41.55±0.02° C. (approximately similar thermal stability to wild-typeAXL) (Table 5).

TABLE 5 Thermal stability of wild-type and mutant AXL proteins asdetermined by variable temperature circular dichroism scans. Average TmIncrease over wt (° C.) +/− (° C.) (° C.) wt AXL 41.27 0.63 — S6-1 54.010.86 12.73 S6-2 41.55 0.02 0.28

TABLE 2 AXL Ig1 mutants from Sorts 5 and 6 (141 total random clonessequenced, 25 unique variants) Clone bp AA 19 23 26 27 32 33 38 44 61 6572 74 78 79 86 87 88 wt AXL A T E E G N T T H D A S O V Q D D AXL S6-1 64 S G AXL S6-2 5 4 G M AXL S6-5 5 4 S N G AXL S5-1 3 3 V AXL S5-2 2 1AXL S5-4 1 1 E AXL S5-6 1 1 V AXL S5-9 4 3 R AXL S5-13 3 2 V AXL S5-22 22 N G AXL S5-24 4 2 G AXL S5-29 9 6 K Y V N AXL S5-30 4 2 AXL S5-39 10 5A V AXL S5-40 3 1 AXL S5-45 5 4 AXL S5-51 3 2 AXL S5-53 3 2 G G AXLS5-59 3 2 I AXL S5-66 2 1 G AXL S5-68 5 2 M AXL S5-74 2 2 V AXL S5-76 2R AXL S5-77 4 T G G AXL S5-78 2 # of Clone 90 92 97 98 105 109 112 113116 118 127 129 repeats wt AXL I V I T T Q V F H T G E AXL S6-1 A R 62AXL S6-2 A E 21 AXL S6-5 A 1 AXL S5-1 R R 1 AXL S5-2 A 16 AXL S5-4 1 AXLS5-6 7 AXL S5-9 V A 10 AXL S5-13 D 1 AXL S5-22 1 AXL S5-24 A 2 AXL S5-29A A 1 AXL S5-30 A R 1 AXL S5-39 V M K 1 AXL S5-40 G 2 AXL S5-45 A A L A1 AXL S5-51 A P 1 AXL S5-53 1 AXL S5-59 A 2 AXL S5-66 3 AXL S5-68 A 1AXL S5-74 L 1 AXL S5-76 A 1 AXL S5-77 A 1 AXL S5-78 M A 1 TOTAL READS:141 *bp = number of DNA mismatches, AA = number of amino acid mutations.Note some of the DNA mutations are silent. Total number of times aparticular clone showed up is indicated in the right most column.

Example 3 Soluble Axl Variants Inhibit Metastatic Tumor Progression InVivo

GAS6-AXL signaling has been implicated in the progression of manyaggressive forms of solid tumors including breast, lung, and colon andrecently through work presented here, ovarian cancer. While a distinctcorrelation has been observed between AXL expression and disease stageand patient prognosis, validation of AXL as a therapeutic target for thetreatment of metastasis has largely remained unexplored. In Example 1,we show that AXL is indeed a marker of metastasis in human breast andovarian cancer patients, with AXL expression levels on primary tumorscorrelating with the severity of the disease. These results suggestedthat antagonizing the GAS6-AXL signaling pathway may offer a therapeuticwindow for treating metastatic disease. As outlined in Example 1, tovalidate the potential of AXL as a therapeutic target, a soluble form ofthe wild-type extracellular domain of AXL was administered usingadenoviral delivery in an aggressive mouse model of human ovariancancer. We showed that tumor metastases were significantly reduced inmice which received the soluble AXL treatments as compared to controls.These data demonstrated that antagonizing GAS6-AXL signaling in tumorcells using soluble AXL could inhibit the metastatic progression of thedisease. Building upon these results, we showed that engineered AXLmutants with higher affinity to GAS6 elicited greater efficacy asanti-metastatic agents, and that a more therapeutically-relevant mode ofdelivering soluble AXL still yielded significant results.

In this study, we used the same human ovarian cancer model outlined inExperiment 1 and administered purified soluble AXL (sAXL) variantsintraperitonealy to mice with pre-existing metastatic disease. We testedboth wild-type AXL and AXL S6-1, the engineered high affinity mutant,and compared both to a form of AXL, E59R/T77R, in which GAS6 binding isabolished. Our results strikingly show that the enhanced affinity of AXLS6-1 results in greater therapeutic efficacy, as a reduction in tumorburden as assessed by both number and total weight of all metastaticlesions was significantly reduced over both wild-type AXL and thenegative control of AXL E59R/T77R. These findings further validate AXLand GAS6 as therapeutic targets for the inhibition of metastasis andsupport the engineered high affinity AXL mutant S6-1 as a potentantagonist of the GAS6-AXL signaling system.

While Example 1 demonstrates that adenoviral delivery of sAXL yieldedtherapeutic efficacy, this method of delivery is not clinically relevantand thus we confirmed that delivery of purified, sAXL would yieldsimilar results. Wild-type AXL, AXL S6-1 and AXL E59R/T77R were fused tothe fragment crystallizable region (Fc) of a mouse IgG2a in order toimprove pharmacokinetics. The only differences between these three AXLfusion (AXL-Fc) variants are mutations found in the AXL Ig1 domain,which are outlined in Table 6A. DNA encoding the AXL-Fc proteins wascloned into the CMV-driven pADD2 adenoviral shuttle vector using EcoRIand SalI restriction sites. The pADD2 plasmid encoding these three AXLmutants was independently transfected into HEK 293 cells using theFreestyle Expression kit from Life Technologies, as described by themanufacturer. Proteins were purified from culture supernatant usingProtein A affinity chromatography followed by size exclusionchromatography.

TABLE 6 Protein name Description Wild-type AXL-Fc Wild-type AXLextracellular domain, amino acids 19-440 fused to the Fc region of mouseIgG2a. AXL S6-1-Fc AXL-Fc fusion as above for wild-type AXL-Fc, however,the AXL Ig1 domain contains the following mutations for S6-1: G32S,D87G, V92A, G127R AXL E59R/T77R-Fc AXL-Fc fusion as above for wild-typeAXL-Fc, however, the Axl Ig1 domain contains E59R and T77R mutations,which significantly diminish binding towards Gas6

To assess the ability of the AXL-Fc mutants to inhibit metastasis invivo, we used the same peritoneal xenograft model of human ovariancancer as outlined in Example 1. This model recapitulates the peritonealdissemination of human ovarian cancer metastasis as mice rapidly develophighly invasive disease consisting of ascites and many (>100) smallmetastatic lesions four weeks post-administration of SKOV3ip.1 cells.This model is a very accurate representation of human ovarian cancer asmost patients present with significant metastatic disease at diagnosis.Mice were injected with SKOV3ip.1 cells and tumors were allowed to seedfor seven days. On day seven, we randomly split the mice into threestudy groups and began administering treatments of either wild-typeAXL-Fc, 56-1-Fc or E59R/T77R-Fc. Purified proteins dissolved inphosphate buffered saline were administered to the mice twice a week forthree weeks at a dose of 10 mg/kg, for a total of six doses. On daytwenty-eight, all mice were sacrificed and necropsies were performed toassess overall tumor burden as measured by the number of visiblemetastatic lesions as well as the total weight of all lesions. Therewere profound differences between the treatment groups, andrepresentative images are shown in FIG. 14. Mice receiving the negativecontrol treatment of E59R/T77R-Fc had an average of 86.3±21.9 peritonealmetastases. For mice receiving wild-type AXL-Fc, that number was reducedto 48.1±6.9 while for mice in the engineered AXL group, S6-1-Fc, only8.3±1.6 metastatic lesions were observed on average (FIG. 15 (toppanel)). All visible lesions were excised and collectively weighed foreach mouse to assess overall metastatic tumor burden. The engineered AXLtreatment group (S6-1-Fc) again showed the most profound response, asE59R/T77R-Fc, wild-type-Fc and S6-1-Fc treatment groups exhibited tumorburdens of 567±92, 430±36 and 188±55 mg, respectively, FIG. 15 (bottompanel).

Collectively, these findings further validate AXL as a therapeutictarget for the treatment of metastasis and demonstrate that neutralizingAXL's ligand, GAS6, is an effective anti-metastatic treatment strategy.Importantly, a protein comprising an AXL-Fc fusion that does not exhibitdetectable binding to Gas6 (AXL E59R/T77R-Fc) does not prevent tumormetastasis; a protein comprising an AXL-Fc fusion that binds to Gas6with moderate affinity (wild-type AXL-Fc) shows slight inhibition oftumor metastasis; a protein comprising an AXL-Fc fusion with very strongaffinity to Gas6 (AXL S6-1-Fc) shows significant inhibition of tumormetastasis. Collectively, this shows that the epitope of interaction forGas6 and AXL is critical in tumor metastasis and potent inhibition ofthis epitope on Gas6 through the AXL S6-1-Fc protein significantlyinhibits tumor metastasis. As such, the AXL S6-1-Fc protein, or anyprotein that potently blocks the Gas6-Axl interaction, is a promisingtherapeutic candidate for metastatic disease. In addition, we alsodemonstrate that direct administration of purified soluble AXL proteinis a viable treatment method, validating this approach clinically.

Methods for Example 3

Cell lines. Ovarian SKOV3ip.1 were cultured in the appropriate mediatesupplemented with 10% fetal bovine serum and 1% penicillin andstreptomycin at 37° C. in a 5% CO₂ incubator.

AXL-Fc fusions. Full-length AXL mutants, amino acids 19-440, were clonedinto the CMV-driven pADD2 adenoviral shuttle vector as direct fusions toa mouse IgG2a Fc region. Transient DNA transfection of human embryonickidney (HEK) 293 cells was accomplished using the Freestyle Expressionkit from Life Technologies, as described by the manufacturer. Fc-fusionproteins were purified from the culture supernatant after five daysusing Protein A affinity chromatography and size exclusionchromatography. Purified proteins were placed in a phosphate bufferedsaline solution without any additional additives or carriers.

SKOV3ip.1 Peritoneal Xenographs. All procedures involving animals andtheir care were approved by the Institutional Animal Care and UsageCommittee of Stanford University in accordance with institutional andNIH guidelines. Six week old female nude mice were injected with 1×10⁶SKOV3ip.1 cells intraperitonealy. Seven days after the administration ofcells, mice were randomly divided into three groups for treatment with56-1-Fc, wild-type AXL-Fc or E59R/T77R-Fc. Purified soluble AXL-Fcprotein was administered via intraperitonealy injections twice a week ata dosage of 10 mg/kg. Dosing was continued for three weeks after whichmice were sacrificed. Necropsies were performed in which metastaticlesions were counted and then excised to be collectively weighted. Tumorburden was determined by both the total number lesions and overallweight of all diseased tissue for each mouse.

Statistical Analysis: Student's t test was used and errors reported arestandard error of the mean (SEM). Values with a p value of <0.01 wereconsidered significant.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

The invention claimed is:
 1. A method of inhibiting growth, metastasisor invasion of a tumor that expresses Axl or GAS6 in a human patient,the method comprising: administering to said patient an effective doseof an isolated soluble AXL variant polypeptide, wherein said polypeptidelacks the AXL transmembrane domain and has a set of amino acidsubstitution(s) of the wild-type AXL sequence (SEQ ID NO. 1) selectedfrom the group consisting of 1) Gly32Ser, Asp87Gly, Val92Ala, andGly127Arq, 2) Glu26Gly, Val79Met, Val92Ala, and Gly127Glu, 3) Asn33Ser,Ser74Asn, Asp87Gly, and Val92Ala, 4) Ala72Val, Ile97Arq, and His116Arq,5) Gln78Glu, 6) Ala72Val, 7) Gln86Arq, Ile90Val, and Val92Ala, 8)Ala72Val, and Val92Asp, 9) Asp65Asn, and Asp87Gly, 10) Asp87Gly, andVal92Ala, 11) Glu27Lys, His61Tyr, Ala72Val, Asp88Asn, Val92Ala, andThr98Ala, 12) Val92Ala, Gln109Arq, 13) Thr44Ala, Ala72Val, Ile90Val,Thr105Met, and Glu129Lys, 14) Val92Gly, 15) Val92Ala, Val112Ala,Phe113Leu, and Thr118Ala, 16) Val92Ala, and Thr98Pro, 17) Glu27Gly, andAsp87Gly, 18) Thr3811e, and Val92Ala, 19) Asp87Gly, 20) Thr23Met, andVal92Ala, 21) Ala72Val, and Phe113Leu, 22) Gln86Arg, Val92Ala, 23)Ala19Thr, Glu26Gly, Glu27Gly, and Val92Ala, 24) Ile90Met and Val92Ala,25) Gly32Ser, and Asp87Gly, 26) Gly32Ser, and Val92Ala, 27) Gly32Ser,and Gly127Arg, 28) Asp87Gly, and Gly127Arg, 29) Val92Ala, and Gly127Arg,30) Asp87Gly, Val92Ala, and Gly127Arg, 31) Gly32Ser, Val92Ala, andGly127Arg, 32) Gly32Ser, Asp87Gly, and Gly127Arg, 33) Gly32Ser,Asp87Gly, and Val92Ala and 34) Gly32Ser, Ala72Val, Asp87Gly, Val92Ala,and Gly127Arg; in a dose effective to inhibit growth, metastasis orinvasion of the tumor that expresses Axl or GAS6.
 2. A method ofinhibiting growth, metastasis or invasion of a tumor that expresses Axlor GAS6 in a human patient, the method comprising: administering anisolated soluble AXL variant polypeptide, wherein said polypeptide lacksthe AXL transmembrane domain and has an amino acid substitution atposition 32, 72, 87, 92, or 127 of the wild-type AXL sequence (SEQ IDNO: 1) or a combination thereof and wherein said polypeptide binds toGAS6 with a binding affinity that is higher than the binding affinity ofthe wild-type AXL to GAS6; in a dose effective to inhibit growth,metastasis or invasion of the tumor that expresses Axl or GAS6.
 3. Themethod of claim 1, wherein the Axl variant polypeptide is a fusionprotein comprising an Fc domain.
 4. The method of claim 2, wherein theAxl variant polypeptide is a fusion protein comprising an Fc domain. 5.The method of claim 1, further comprising administering at least onecytotoxic agent to said patient.
 6. The method of claim 2, furthercomprising administering at least one cytotoxic agent to said patient.